The intestinal microbiome influences health and disease. Its composition is affected by host genetics and environmental exposures. Understanding host genetic effects is critical but challenging in humans, due to the difficulty of detecting, mapping and interpreting them. To address this, we analysed host genetic effects in four cohorts of outbred laboratory rats exposed to distinct but controlled environments. We found that polygenic host genetic effects were consistent across environments. We identified three replicated microbiome-associated loci, one of which involved a sialyltransferase gene and Paraprevotella. We found a similar association in a human cohort, between ST6GAL1 and Paraprevotella, both of which have been linked with immune and infectious diseases. Moreover, we found evidence of indirect genetic effects on microbiome phenotypes, which substantially increased their total genetic variance. Finally, we identified a novel mechanism whereby indirect genetic effects can contribute to "missing heritability".

The intestinal microbiome influences health and disease. Its composition is affected by host genetics and environmental exposures. Understanding host genetic effects is critical but challenging in humans, due to the difficulty of detecting, mapping and interpreting them. To address this, we analyse host genetic effects in four cohorts of outbred laboratory rats exposed to distinct but controlled environments. We show that polygenic host genetic effects are consistent across cohort environments. We identify three replicated microbiome-associated loci, one of which involves the sialyltransferase gene St6galnac1 and Paraprevotella. We find a similar association in a human cohort, between ST6GAL1 and Paraprevotella, both of which have been linked with immune and infectious diseases. Moreover, we find indirect (i.e. social) genetic effects on microbiome phenotypes, which substantially increase the total genetic variance. Finally, we identify a novel mechanism whereby indirect genetic effects can contribute to “missing heritability”. The intestinal microbiome is shaped by genetics and environment. Here, the authors show in rats that host genetic effects, including indirect social effects, influence microbiome composition, identify replicated loci, and reveal mechanisms contributing to microbiome heritability.

Genome-wide association studies (GWAS) have implicated specific alleles and genes as risk factors for numerous complex traits. However, translating GWAS results into biologically and therapeutically meaningful discoveries remains extremely challenging. Most GWAS results identify noncoding regions of the genome, suggesting that differences in gene regulation are the major driver of trait variability. To better integrate GWAS results with gene regulatory polymorphisms, we previously developed PrediXcan (also known as "transcriptome-wide association studies" or TWAS), which maps SNPs to predicted gene expression using GWAS data. In this study, we developed RatXcan, a framework that extends this methodology to outbred heterogeneous stock (HS) rats. RatXcan accounts for the close familial relationships among HS rats by modeling the relatedness with a random effect that encodes the genetic relatedness. RatXcan also corrects for polygenic-driven inflation because of the equivalence between a relatedness random effect and the infinitesimal polygenic model. To develop RatXcan, we trained transcript predictors for 8,934 genes using reference genotype and expression data from five rat brain regions. We found that the cis genetic architecture of gene expression in both rats and humans was sparse and similar across brain tissues. We tested the association between predicted expression in rats and two example traits (body length and BMI) using phenotype and genotype data from 5,401 densely genotyped HS rats and identified a significant enrichment between the genes associated with rat and human body length and BMI. Thus, RatXcan represents a valuable tool for identifying the relationship between gene expression and phenotypes across species and paves the way to explore shared biological mechanisms of complex traits.

Genome-wide association studies typically evaluate the autosomes and sometimes the X Chromosome, but seldom consider the Y or mitochondrial (MT) Chromosomes. We genotyped the Y and MT Chromosomes in heterogeneous stock (HS) rats (Rattus norvegicus), an outbred population created from 8 inbred strains. We identified 8 distinct Y and 4 distinct MT Chromosomes among the 8 founders. However, only 2 types of each nonrecombinant chromosome were observed in our modern HS rat population (generations 81-97). Despite the relatively large sample size, there were virtually no significant associations for behavioral, physiological, metabolome, or microbiome traits after correcting for multiple comparisons. However, both Y and MT Chromosomes were strongly associated with the expression of a few genes located on those chromosomes, which provided a positive control. Our results suggest that within modern HS rats there are no Y and MT Chromosomes differences that strongly influence behavioral or physiological traits. These results do not address other ancestral Y and MT Chromosomes that do not appear in modern HS rats, nor do they address effects that may exist in other rat populations, or in other species.

Elevated intraocular pressure (IOP) is influenced by environmental and genetic factors. Increased IOP is a major risk factor for most types of glaucoma, including primary open angle glaucoma (POAG). Investigating the genetic basis of IOP may lead to a better understanding of the molecular mechanisms of POAG. The goal of this study was to identify genetic loci involved in regulating IOP using outbred heterogeneous stock (HS) rats. HS rats are a multigenerational outbred population derived from eight inbred strains that have been fully sequenced. This population is ideal for a genome-wide association study (GWAS) owing to the accumulated recombinations among well-defined haplotypes, the relatively high allele frequencies, the accessibility to a large collection of tissue samples, and the large allelic effect size compared to human studies. Both male and female HS rats (N = 1,812) were used in the study. Genotyping-by-sequencing was used to obtain ∼3.5 million single nucleotide polymorphisms (SNP) from each individual. SNP heritability for IOP in HS rats was 0.32, which agrees with other studies. We performed a GWAS for the IOP phenotype using a linear mixed model and used permutation to determine a genome-wide significance threshold. We identified three genome-wide significant loci for IOP on chromosomes 1, 5, and 16. Next, we sequenced the mRNA of 51 whole eye samples to find cis-eQTLs to aid in identification of candidate genes. We report 5 candidate genes within those loci: Tyr, Ctsc, Plekhf2, Ndufaf6 and Angpt2. Tyr, Ndufaf6 and Angpt2 genes have been previously implicated by human GWAS of IOP-related conditions. Ctsc and Plekhf2 genes represent novel findings that may provide new insight into the molecular basis of IOP. This study highlights the efficacy of HS rats for investigating the genetics of elevated IOP and identifying potential candidate genes for future functional testing.

Young blood plasma is known to confer beneficial effects on various organs in mice and rats. However, it was not known whether plasma from young adult pigs rejuvenates old rat tissues at the epigenetic level; whether it alters the epigenetic clock, which is a highly accurate molecular biomarker of aging. To address this question, we developed and validated six different epigenetic clocks for rat tissues that are based on DNA methylation values derived from n = 613 tissue samples. As indicated by their respective names, the rat pan-tissue clock can be applied to DNA methylation profiles from all rat tissues, while the rat brain, liver, and blood clocks apply to the corresponding tissue types. We also developed two epigenetic clocks that apply to both human and rat tissues by adding n = 1366 human tissue samples to the training data. We employed these six rat clocks to investigate the rejuvenation effects of a porcine plasma fraction treatment in different rat tissues. The treatment more than halved the epigenetic ages of blood, heart, and liver tissue. A less pronounced, but statistically significant, rejuvenation effect could be observed in the hypothalamus. The treatment was accompanied by progressive improvement in the function of these organs as ascertained through numerous biochemical/physiological biomarkers, behavioral responses encompassing cognitive functions. An immunoglobulin G (IgG) N-glycosylation pattern shift from pro- to anti-inflammatory also indicated reversal of glycan aging. Overall, this study demonstrates that a young porcine plasma-derived treatment markedly reverses aging in rats according to epigenetic clocks, IgG glycans, and other biomarkers of aging.

Power analyses are often used to determine the number of animals required for a genome wide association analysis (GWAS). These analyses are typically intended to estimate the sample size needed for at least one locus to exceed a genome-wide significance threshold. A related question that is less commonly considered is the number of significant loci that will be discovered with a given sample size. We used simulations based on a real dataset that consisted of 3,173 male and female adult N/NIH heterogeneous stock (HS) rats to explore the relationship between sample size and the number of significant loci discovered. Our simulations examined the number of loci identified in sub-samples of the full dataset. The sub-sampling analysis was conducted for four traits with low (0.15 ± 0.03), medium (0.31 ± 0.03 and 0.36 ± 0.03) and high (0.46 ± 0.03) SNP-based heritabilities. For each trait, we sub-sampled the data 100 times at different sample sizes (500, 1,000, 1,500, 2,000, and 2,500). We observed an exponential increase in the number of significant loci with larger sample sizes. Our results are consistent with similar observations in human GWAS and imply that future rodent GWAS should use sample sizes that are significantly larger than those needed to obtain a single significant result.

1 Abstract Human unilateral renal agenesis is a congenital urinary tract malformation. Affected individuals have only one kidney, which is often an asymptomatic developmental defect. A total of 5,585 male and female HS rats were assessed for unilateral renal agenesis and genotyped for 3’513,321 markers. The R package SAIGEgds was used for the association analysis. The adjusted p-value threshold for the association analysis determined by permutation was equal to 5.6 (-log10). Two additional datasets were used as validation tests. Population two included 1,577 rats genotyped for 7,425,889 markers and a case-control imbalance equal to 1:174; population three included 1,407 rats, genotyped for 254,932 markers and case-control ratio equal to 1:38. The python package GxTheta was used to perform a polygenic epistasis analysis for the analyzed HS rat population. A founder haplotype mosaic determination was performed using the R package QTL2. Associated regions were selected for further analysis, including long-read PacBio sequencing for founder individuals and a founder haplotype prediction test. A similarity analysis at a genomic level and for loci encoding transcription factors predicted to interact with selected sequences inside the associated loci were accomplished. A total of 1,181 polymorphisms were associated with URA. All associated polymorphisms were located on chromosome 14 between 32.9 and 36.6 Mb. The most significant polymorphism was chr14:36,411,266, a G/T transversion. The same associated region was identified in population three. Polygenic epistasis was determined as not predominant for the presentation of URA. Based on the haplotype mosaic probability estimation, cases display a higher probability of inheriting the ACI allele. The long-read sequencing analysis showed the presence of an Erv insertion inside the intron one of the KIT gene located inside the associated region. The Erv insertion comprises one Erv sequence and two Ltr sequences located downstream and upstream of the former. No Erv insertion was identified for the founder strain BN. For ACI and HSRA, only one Ltr sequence was identified. One hundred and seven genes encoding TFs that recognize binding sites on the Erv insertion were analyzed for sequence similarity against the reference HSRA. The TF similarity score analysis for the interaction genotype and phenotype showed significance after FDR correction for 20 TFs, including AHR, HNF1B, JUNB, RARG, and RXRA. A mechanism identifying URA as a threshold phenotype is suggested in HS rats. It implies the existence of a minimum threshold for the final number of nephrons and kidney associated structures required for stalling the apoptotic process of the metanephric rudiments. Animals exhibiting a quantitative cumulative defect would express URA, being this malformation identified as a phenotype with decreased penetrance in the assessed population of HS rats. All these processes are described as mediated by KIT and TFs able to interact with sequences of the Erv insertion.

A vexing observation in genome-wide association studies (GWASs) is that parallel analyses in different species may not identify orthologous genes. Here, we demonstrate that cross-species translation of GWASs can be greatly improved by an analysis of co-localization within molecular networks. Using body mass index (BMI) as an example, we show that the genes associated with BMI in humans lack significant agreement with those identified in rats. However, the networks interconnecting these genes show substantial overlap, highlighting common mechanisms including synaptic signaling, epigenetic modification, and hormonal regulation. Genetic perturbations within these networks cause abnormal BMI phenotypes in mice, too, supporting their broad conservation across mammals. Other mechanisms appear species specific, including carbohydrate biosynthesis (humans) and glycerolipid metabolism (rodents). Finally, network co-localization also identifies cross-species convergence for height/body length. This study advances a general paradigm for determining whether and how phenotypes measured in model species recapitulate human biology.

Quantitative Trait Locus (QTL) analysis and Genome-Wide Association Studies (GWAS) have the power to identify variants that capture significant levels of phenotypic variance in complex traits. However, effort and time are required to select the best methods and optimize parameters and pre-processing steps. Although machine learning approaches have been shown to greatly assist in optimization and data processing, applying them to QTL analysis and GWAS is challenging due to the complexity of large, heterogenous datasets. Here, we describe proof-of-concept for an automated machine learning approach, AutoQTL, with the ability to automate many complicated decisions related to analysis of complex traits and generate solutions to describe relationships that exist in genetic data. Using a publicly available dataset of 18 putative QTL from a large-scale GWAS of body mass index in the laboratory rat, Rattus norvegicus, AutoQTL captures the phenotypic variance explained under a standard additive model. AutoQTL also detects evidence of non-additive effects including deviations from additivity and 2-way epistatic interactions in simulated data via multiple optimal solutions. Additionally, feature importance metrics provide different insights into the inheritance models and predictive power of multiple GWAS-derived putative QTL. This proof-of-concept illustrates that automated machine learning techniques can complement standard approaches and have the potential to detect both additive and non-additive effects via various optimal solutions and feature importance metrics. In the future, we aim to expand AutoQTL to accommodate omics-level datasets with intelligent feature selection and feature engineering strategies.

Interpreting and integrating results from omics studies typically requires a comprehensive and time consuming survey of extant literature. GeneCup is a literature mining web service that retrieves sentences containing user-provided gene symbols and keywords from PubMed abstracts. The keywords are organized into an ontology and can be extended to include results from human genome-wide association studies. We provide a drug addiction keyword ontology that contains over 300 keywords as an example. The literature search is conducted by querying the PubMed server using a programming interface, which is followed by retrieving abstracts from a local copy of the PubMed archive. The main results presented to the user are sentences where gene symbol and keywords co-occur. These sentences are presented through an interactive graphical interface or as tables. All results are linked to the original abstract in PubMed. In addition, a convolutional neural network is employed to distinguish sentences describing systemic stress from those describing cellular stress. The automated and comprehensive search strategy provided by GeneCup facilitates the integration of new discoveries from omic studies with existing literature. GeneCup is free and open source software. The source code of GeneCup and the link to a running instance is available at https://github.com/hakangunturkun/GeneCup.

Genome-wide association studies have demonstrated that most 35 traits are highly polygenic; however, translating these polygenic signals into 36 biological insights remains difficult. A lack of satisfactory methods for 37 translating polygenic results across species has precluded the use of model 38 organisms to address this problem. Here we explore the use of polygenic 39 transcriptomic risk scores (PTRS) for translating polygenic results across species. 40 Unlike polygenic risk scores (PRS), which rely on SNPs for predicting traits, PTRS 41 use imputed gene expression for prediction, which allows cross-species 42 translation to orthologous genes. We first developed RatXcan, which is a 43 framework for transcriptome-wide association studies (TWAS) in outbred rats. 44 Leveraging predicted transcriptome and genotype data from UK Biobank, and 45 the genetically trained gene expression models from RatXcan, we scored more 46 than 3,000 rats using a human-derived PTRS for height. Strikingly, we found that 47 human-derived height PTRS significantly predicted body length in rats (P<0.013). 48 The genes included in the PTRS were enriched for biological pathways including 49 skeletal growth and metabolism and were over-represented in tissues including 50 pancreas and brain. This approach facilitates experimental studies in model 51 organisms that examine the polygenic basis of human complex traits and 52 provides an empirical metric by which to evaluate the suitability of specific 53 animal models and identify their shared biological underpinnings. 54

Many personality traits are influenced by genetic factors. Rodents models provide an efficient system for analyzing genetic contribution to these traits. Using 1,246 adolescent heterogeneous stock (HS) male and female rats, we conducted a genome-wide association study (GWAS) of behaviors measured in an open field, including locomotion, novel object interaction, and social interaction. We identified 30 genome-wide significant quantitative trait loci (QTL). Using multiple criteria, including the presence of high impact genomic variants and co-localization of cis-eQTL, we identified 17 candidate genes (Adarb2, Ankrd26, Cacna1c, Cacng4, Clock, Ctu2, Cyp26b1, Dnah9, Gda, Grxcr1, Eva1a, Fam114a1, Kcnj9, Mlf2, Rab27b, Sec11a, and Ube2h) for these traits. Many of these genes have been implicated by human GWAS of various psychiatric or drug abuse related traits. In addition, there are other candidate genes that likely represent novel findings that can be the catalyst for future molecular and genetic insights into human psychiatric diseases. Together, these findings provide strong support for the use of the HS population to study psychiatric disorders.

Heterogeneous Stock (HS) rats are a genetically diverse outbred rat population that is widely used for studying genetics of behavioral and physiological traits. Mapping Quantitative Trait Loci (QTL) associated with transcriptional changes would help to identify mechanisms underlying these traits. We generated genotype and transcriptome data for five brain regions from 88 HS rats. We identified 21 392 cis-QTLs associated with expression and splicing changes across all five brain regions and validated their effects using allele specific expression data. We identified 80 cases where eQTLs were colocalized with genome-wide association study (GWAS) results from nine physiological traits. Comparing our dataset to human data from the Genotype-Tissue Expression (GTEx) project, we found that the HS rat data yields twice as many significant eQTLs as a similarly sized human dataset. We also identified a modest but highly significant correlation between genetic regulatory variation among orthologous genes. Surprisingly, we found less genetic variation in gene regulation in HS rats relative to humans, though we still found eQTLs for the orthologs of many human genes for which eQTLs had not been found. These data are available from the RatGTEx data portal (RatGTEx.org) and will enable new discoveries of the genetic influences of complex traits.

Vincristine (VCR) is the first-line chemotherapeutic medication often co-administered with other drugs to treat childhood acute lymphoblastic leukemia. Dose-dependent neurotoxicity is the main factor restricting VCR's clinical application. VCR-induced peripheral neuropathy (VIPN) sometimes results in dose reduction or omission, leading to clinical complications or affecting the patient's quality of life. With regard to the genetic basis of drug responses, preemptive pharmacogenomic testing and simultaneous blood level monitoring could be helpful for the transformation of various findings into individualized therapies. In this review, we discussed the potential associations between genetic variants in genes contributing to the pharmacokinetics/pharmacodynamics of VCR and VIPN incidence and severity in patients with acute lymphoblastic leukemia. Of note, genetic variants in the CEP72 gene have great potential to be translated into clinical practice. Such a genetic biomarker may help clinicians diagnose VIPN earlier. Besides, genetic variants in other genes, such as CYP3A5, ABCB1, ABCC1, ABCC2, TTPA, ACTG1, CAPG, SYNE2, SLC5A7, COCH, and MRPL47, have been reported to be associated with the VIPN, but more evidence is needed to validate the findings in the future. In fact, a variety of complex factors jointly determine the VIPN. In implementing precision medicine, the combination of genetic, environmental, and personal variables, along with therapeutic drug monitoring, will allow for a better understanding of the mechanisms of VIPN, improving the effectiveness of VCR treatment, reducing adverse reactions, and improving patients' quality of life.

In this study, we investigated whether intravenously self-administered nicotine with menthol and audiovisual cue modulates nicotine-metabolizing CYP2A6, oxidative stress modulators, and cytokines/chemokines in plasma extracellular vesicles (EVs) in rats. We assigned rats to self-administered nicotine with: (a) audiovisual cue (AV), (b) menthol, and (c) menthol and AV cue. We found increased levels of CD9 in plasma EVs after self-administered nicotine with menthol and AV cue. Moreover, expression of CYP2A6 in plasma EVs was significantly increased after self-administered nicotine in response to menthol and AV cue. However, despite an upward trend on SOD1 and catalase, increase was not found to be statistically significant, while total antioxidant capacity was found to be significantly increased in plasma and plasma EVs obtained after self-administered nicotine with menthol and AV cue. Among cytokine and chemokine profiling, we found a significant increase in the levels of MCP-1 after self-administered nicotine with menthol and AV cue and complete packaging of IL-1β in EVs. Taken together, the study provides evidence that nicotine in response to menthol and AV cues can package altered levels of CYP2A6, and cytokines/chemokines in plasma EVs that may contribute to cell-cell communication, nicotine metabolism, and inflammation upon cigarette smoking.

Green tea (Camellia sinensis, Theaceace), has been extensively studied for its putative effects in prevention of age related diseases. Here, we discuss the increasing evidence that consumption of green tea has preventative effects in obesity, hypertension, insulin resistance, type II diabetes, atherosclerosis, coronary heart disease and Metabolic Syndrome (MetS). The catechins in green tea has been found to be beneficial in obesity induced by a high-fat diet. These effects are mainly attributable to the gallate esters of catechins, (-)-epicatechin gallate (ECG) and (-)-epigallocatechin-3- gallate (EGCG).

Young blood plasma is known to confer beneficial effects on various organs in mice. However, it was not known whether young plasma rejuvenates cells and tissues at the epigenetic level; whether it alters the epigenetic clock, which is a highly-accurate molecular biomarker of aging. To address this question, we developed and validated six different epigenetic clocks for rat tissues that are based on DNA methylation values derived from n=593 tissue samples. As indicated by their respective names, the rat pan-tissue clock can be applied to DNA methylation profiles from all rat tissues, while the rat brain-, liver-, and blood clocks apply to the corresponding tissue types. We also developed two epigenetic clocks that apply to both human and rat tissues by adding n=850 human tissue samples to the training data. We employed these six clocks to investigate the rejuvenation effects of a plasma fraction treatment in different rat tissues. The treatment more than halved the epigenetic ages of blood, heart, and liver tissue. A less pronounced, but statistically significant, rejuvenation effect could be observed in the hypothalamus. The treatment was accompanied by progressive improvement in the function of these organs as ascertained through numerous biochemical/physiological biomarkers and behavioral responses to assess cognitive functions. Cellular senescence, which is not associated with epigenetic aging, was also considerably reduced in vital organs. Overall, this study demonstrates that a plasma-derived treatment markedly reverses aging according to epigenetic clocks and benchmark biomarkers of aging.