A susceptibility gene signature for ERBB2-driven mammary tumour development and metastasis in collaborative cross mice.

BACKGROUND: Deeper insights into ERBB2-driven cancers are essential to develop new treatment approaches for ERBB2+ breast cancers (BCs). We employed the Collaborative Cross (CC) mouse model to unearth genetic factors underpinning Erbb2-driven mammary tumour development and metastasis. METHODS: 732 F1 hybrid female mice between FVB/N MMTV-Erbb2 and 30 CC strains were monitored for mammary tumour phenotypes. GWAS pinpointed SNPs that influence various tumour phenotypes. Multivariate analyses and models were used to construct the polygenic score and to develop a mouse tumour susceptibility gene signature (mTSGS), where the corresponding human ortholog was identified and designated as hTSGS. The importance and clinical value of hTSGS in human BC was evaluated using public datasets, encompassing TCGA, METABRIC, GSE96058, and I-SPY2 cohorts. The predictive power of mTSGS for response to chemotherapy was validated in vivo using genetically diverse MMTV-Erbb2 mice. FINDINGS: Distinct variances in tumour onset, multiplicity, and metastatic patterns were observed in F1-hybrid female mice between FVB/N MMTV-Erbb2 and 30 CC strains. Besides lung metastasis, liver and kidney metastases emerged in specific CC strains. GWAS identified specific SNPs significantly associated with tumour onset, multiplicity, lung metastasis, and liver metastasis. Multivariate analyses flagged SNPs in 20 genes (Stx6, Ramp1, Traf3ip1, Nckap5, Pfkfb2, Trmt1l, Rprd1b, Rer1, Sepsecs, Rhobtb1, Tsen15, Abcc3, Arid5b, Tnr, Dock2, Tti1, Fam81a, Oxr1, Plxna2, and Tbc1d31) independently tied to various tumour characteristics, designated as a mTSGS. hTSGS scores (hTSGSS) based on their transcriptional level showed prognostic values, superseding clinical factors and PAM50 subtype across multiple human BC cohorts, and predicted pathological complete response independent of and superior to MammaPrint score in I-SPY2 study. The power of mTSGS score for predicting chemotherapy response was further validated in an in vivo mouse MMTV-Erbb2 model, showing that, like findings in human patients, mouse tumours with low mTSGS scores were most likely to respond to treatment. INTERPRETATION: Our investigation has unveiled many new genes predisposing individuals to ERBB2-driven cancer. Translational findings indicate that hTSGS holds promise as a biomarker for refining treatment strategies for patients with BC. FUNDING: The U.S. Department of Defense (DoD) Breast Cancer Research Program (BCRP) (BC190820), United States; MCIN/AEI/10.13039/501100011039 (PID2020-118527RB-I00, PDC2021-121735-I00), the "European Union Next Generation EU/PRTR," the Regional Government of Castile and León (CSI144P20), European Union.

Studying the Effect of the Host Genetic Background of Juvenile Polyposis Development Using Collaborative Cross and Smad4 Knock-Out Mouse Models.

Juvenile polyposis syndrome (JPS) is a rare autosomal dominant disorder characterized by multiple juvenile polyps in the gastrointestinal tract, often associated with mutations in genes such as Smad4 and BMPR1A. This study explores the impact of Smad4 knock-out on the development of intestinal polyps using collaborative cross (CC) mice, a genetically diverse model. Our results reveal a significant increase in intestinal polyps in Smad4 knock-out mice across the entire population, emphasizing the broad influence of Smad4 on polyposis. Sex-specific analyses demonstrate higher polyp counts in knock-out males and females compared to their WT counterparts, with distinct correlation patterns. Line-specific effects highlight the nuanced response to Smad4 knock-out, underscoring the importance of genetic variability. Multimorbidity heat maps offer insights into complex relationships between polyp counts, locations, and sizes. Heritability analysis reveals a significant genetic basis for polyp counts and sizes, while machine learning models, including k-nearest neighbors and linear regression, identify key predictors, enhancing our understanding of juvenile polyposis genetics. Overall, this study provides new information on understanding the intricate genetic interplay in the context of Smad4 knock-out, offering valuable insights that could inform the identification of potential therapeutic targets for juvenile polyposis and related diseases.

Genetic and microbial determinants of azoxymethane-induced colorectal tumor susceptibility in Collaborative Cross mice and their implication in human cancer.

The insights into interactions between host genetics and gut microbiome (GM) in colorectal tumor susceptibility (CTS) remains lacking. We used Collaborative Cross mouse population model to identify genetic and microbial determinants of Azoxymethane-induced CTS. We identified 4417 CTS-associated single nucleotide polymorphisms (SNPs) containing 334 genes that were transcriptionally altered in human colorectal cancers (CRCs) and consistently clustered independent human CRC cohorts into two subgroups with different prognosis. We discovered a set of genera in early-life associated with CTS and defined a 16-genus signature that accurately predicted CTS, the majority of which were correlated with human CRCs. We identified 547 SNPs associated with abundances of these genera. Mediation analysis revealed GM as mediators partially exerting the effect of SNP UNC3869242 within Duox2 on CTS. Intestine cell-specific depletion of Duox2 altered GM composition and contribution of Duox2 depletion to CTS was significantly influenced by GM. Our findings provide potential novel targets for personalized CRC prevention and treatment.

Single-Cell Transcriptomics of Bone Marrow Stromal Cells in Diversity Outbred Mice: A Model for Population-Level scRNA-Seq Studies.

Genome-wide association studies (GWASs) have advanced our understanding of the genetics of osteoporosis; however, the challenge has been converting associations to causal genes. Studies have utilized transcriptomics data to link disease-associated variants to genes, but few population transcriptomics data sets have been generated on bone at the single-cell level. To address this challenge, we profiled the transcriptomes of bone marrow-derived stromal cells (BMSCs) cultured under osteogenic conditions from five diversity outbred (DO) mice using single-cell RNA-seq (scRNA-seq). The goal of the study was to determine if BMSCs could serve as a model to generate cell type-specific transcriptomic profiles of mesenchymal lineage cells from large populations of mice to inform genetic studies. By enriching for mesenchymal lineage cells in vitro, coupled with pooling of multiple samples and downstream genotype deconvolution, we demonstrate the scalability of this model for population-level studies. We demonstrate that dissociation of BMSCs from a heavily mineralized matrix had little effect on viability or their transcriptomic signatures. Furthermore, we show that BMSCs cultured under osteogenic conditions are diverse and consist of cells with characteristics of mesenchymal progenitors, marrow adipogenic lineage precursors (MALPs), osteoblasts, osteocyte-like cells, and immune cells. Importantly, all cells were similar from a transcriptomic perspective to cells isolated in vivo. We employed scRNA-seq analytical tools to confirm the biological identity of profiled cell types. SCENIC was used to reconstruct gene regulatory networks (GRNs), and we observed that cell types show GRNs expected of osteogenic and pre-adipogenic lineage cells. Further, CELLECT analysis showed that osteoblasts, osteocyte-like cells, and MALPs captured a significant component of bone mineral density (BMD) heritability. Together, these data suggest that BMSCs cultured under osteogenic conditions coupled with scRNA-seq can be used as a scalable and biologically informative model to generate cell type-specific transcriptomic profiles of mesenchymal lineage cells in large populations. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Unraveling the Host Genetic Background Effect on Internal Organ Weight Influenced by Obesity and Diabetes Using Collaborative Cross Mice.

Type 2 diabetes mellitus (T2DM) is a severe chronic epidemic that results from the body's improper usage of the hormone insulin. Globally, 700 million people are expected to have received a diabetes diagnosis by 2045, according to the International Diabetes Federation (IDF). Cancer and macro- and microvascular illnesses are only a few immediate and long-term issues it could lead to. T2DM accelerates the effect of organ weights by triggering a hyperinflammatory response in the body's organs, inhibiting tissue repair and resolving inflammation. Understanding how genetic variation translates into different clinical presentations may highlight the mechanisms through which dietary elements may initiate or accelerate inflammatory disease processes and suggest potential disease-prevention techniques. To address the host genetic background effect on the organ weight by utilizing the newly developed mouse model, the Collaborative Cross mice (CC). The study was conducted on 207 genetically different CC mice from 8 CC lines of both sexes. The experiment started with 8-week-old mice for 12 weeks. During this period, one group maintained a standard chow diet (CHD), while the other group maintained a high-fat diet (HFD). In addition, body weight was recorded bi-weekly, and at the end of the study, a glucose tolerance test, as well as tissue collection (liver, spleen, heart), were conducted. Our study observed a strong effect of HFD on blood glucose clearance among different CC lines. The HFD decreased the blood glucose clearance displayed by the significant Area Under Curve (AUC) values in both populations. In addition, variation in body weight changes among the different CC lines in response to HFD. The female liver weight significantly increased compared to males in the overall population when exposed to HFD. Moreover, males showed higher heritability values than females on the same diet. Regardless of the dietary challenge, the liver weight in the overall male population correlated positively with the final body weight. The liver weight results revealed that three different CC lines perform well under classification models. The regression results also varied among organs. Accordingly, the differences among these lines correspond to the genetic variance, and we suspect that some genetic factors invoke different body responses to HFD. Further investigations, such as quantitative trait loci (QTL) analysis and genomic studies, could find these genetic elements. These findings would prove critical factors for developing personalized medicine, as they could indicate future body responses to numerous situations early, thus preventing the development of complex diseases.

Thirdhand tobacco smoke exposure increases the genetic background-dependent risk of pan-tumor development in Collaborative Cross mice.

Increasing evidence has shown that thirdhand smoke (THS) exposure is likely to induce adverse health effects. An important knowledge gap remains in our understanding of THS exposure related to cancer risk in the human population. Population-based animal models are useful and powerful in investigating the interplay between host genetics and THS exposure on cancer risk. Here, we used the Collaborative Cross (CC) mouse population-based model system, which recapitulates the genetic and phenotypic diversity observed in the human population, to assess cancer risk after a short period of exposure, between 4 and 9 weeks of age. Eight CC strains (CC001, CC019, CC026, CC036, CC037, CC041, CC042 and CC051) were included in our study. We quantified pan-tumor incidence, tumor burden per mouse, organ tumor spectrum and tumor-free survival until 18 months of age. At the population level, we observed a significantly increased pan-tumor incidence and tumor burden per mouse in THS-treated mice as compared to the control (p = 3.04E-06). Lung and liver tissues exhibited the largest risk of undergoing tumorigenesis after THS exposure. Tumor-free survival was significantly reduced in THS-treated mice compared to control (p = 0.044). At the individual strain level, we observed a large variation in tumor incidence across the 8 CC strains. CC036 and CC041 exhibited a significant increase in pan-tumor incidence (p = 0.0084 and p = 0.000066, respectively) after THS exposure compared to control. We conclude that early-life THS exposure increases tumor development in CC mice and that host genetic background plays an important role in individual susceptibility to THS-induced tumorigenesis. Genetic background is an important factor that should be taken into account when determining human cancer risk of THS exposure.

Intestinal cancer development in response to oral infection with high-fat diet-induced Type 2 diabetes (T2D) in collaborative cross mice under different host genetic background effects.

Type 2 diabetes (T2D) is a metabolic disease with an imbalance in blood glucose concentration. There are significant studies currently showing association between T2D and intestinal cancer developments. High-fat diet (HFD) plays part in the disease development of T2D, intestinal cancer and infectious diseases through many biological mechanisms, including but not limited to inflammation. Understanding the system genetics of the multimorbidity of these diseases will provide an important knowledge and platform for dissecting the complexity of these diseases. Furthermore, in this study we used some machine learning (ML) models to explore more aspects of diabetes mellitus. The ultimate aim of this project is to study the genetic factors, which underline T2D development, associated with intestinal cancer in response to a HFD consumption and oral coinfection, jointly or separately, on the same host genetic background. A cohort of 307 mice of eight different CC mouse lines in the four experimental groups was assessed. The mice were maintained on either HFD or chow diet (CHD) for 12-week period, while half of each dietary group was either coinfected with oral bacteria or uninfected. Host response to a glucose load and clearance was assessed using intraperitoneal glucose tolerance test (IPGTT) at two time points (weeks 6 and 12) during the experiment period and, subsequently, was translated to area under curve (AUC) values. At week 5 of the experiment, mice of group two and four were coinfected with Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) strains, three times a week, while keeping the other uninfected mice as a control group. At week 12, mice were killed, small intestines and colon were extracted, and subsequently, the polyp counts were assessed; as well, the intestine lengths and size were measured. Our results have shown that there is a significant variation in polyp's number in different CC lines, with a spectrum between 2.5 and 12.8 total polyps on average. There was a significant correlation between area under curve (AUC) and intestine measurements, including polyp counts, length and size. In addition, our results have shown a significant sex effect on polyp development and glucose tolerance ability with males more susceptible to HFD than females by showing higher AUC in the glucose tolerance test. The ML results showed that classification with random forest could reach the highest accuracy when all the attributes were used. These results provide an excellent platform for proceeding toward understanding the nature of the genes involved in resistance and rate of development of intestinal cancer and T2D induced by HFD and oral coinfection. Once obtained, such data can be used to predict individual risk for developing these diseases and to establish the genetically based strategy for their prevention and treatment.

Using the Collaborative Cross and Diversity Outbred Mice in Immunology.

The Collaborative Cross (CC) and the Diversity Outbred (DO) stock mouse panels are the most powerful murine genetics tools available to the genetics community. Together, they combine the strength of inbred animal models with the diversity of outbred populations. Using the 63 CC strains or a panel of DO mice, each derived from the same 8 parental mouse strains, researchers can map genetic contributions to exceptionally complex immunological and infectious disease traits that would require far greater powering if performed by genome-wide association studies (GWAS) in human populations. These tools allow genes to be studied in heterozygous and homozygous states and provide a platform to study epistasis between interacting loci. Most importantly, once a quantitative phenotype is investigated and quantitative trait loci are identified, confirmatory genetic studies can be performed, which is often problematic using the GWAS approach. In addition, novel stable mouse models for immune phenotypes are often derived from studies utilizing the DO and CC mice that can serve as stronger model systems than existing ones in the field. The CC/DO systems have contributed to the fields of cancer immunology, autoimmunity, vaccinology, infectious disease, allergy, tissue rejection, and tolerance but have thus far been greatly underutilized. In this article, we present a recent review of the field and point out key areas of immunology that are ripe for further investigation and awaiting new CC/DO research projects. We also highlight some of the strong computational tools that have been developed for analyzing CC/DO genetic and phenotypic data. Additionally, we have formed a centralized community on the CyVerse infrastructure where immunogeneticists can utilize those software tools, collaborate with groups across the world, and expand the use of the CC and DO systems for investigating immunogenetic phenomena. © 2022 Wiley Periodicals LLC.

A Locus on Chromosome 15 Contributes to Acute Ozone-induced Lung Injury in Collaborative Cross Mice.

Ozone (O3)-induced respiratory toxicity varies considerably within the human population and across inbred mouse strains, indicative of gene-environment interactions (GxE). Though previous studies have identified several quantitative trait loci (QTL) and candidate genes underlying responses to O3 exposure, precise mechanisms of susceptibility remain incompletely described. We sought to update our understanding of the genetic architecture of O3 responsiveness using the Collaborative Cross (CC) recombinant inbred mouse panel. We evaluated hallmark O3-induced inflammation and injury phenotypes in 56 CC strains after exposure to filtered air or 2 ppm O3, and performed focused genetic analysis of variation in lung injury, as reflected by protein in lung lavage fluid. Strain-dependent responses to O3 were clear, and QTL mapping revealed two novel loci on Chr (Chromosomes) 10 (peak, 26.2 Mb; 80% confidence interval [CI], 24.6-43.6 Mb) and 15 (peak, 47.1 Mb; 80% CI, 40.2-54.9 Mb), the latter surpassing the 95% significance threshold. At the Chr 15 locus, C57BL/6J and CAST/EiJ founder haplotypes were associated with higher lung injury responses compared with all other CC founder haplotypes. With further statistical analysis and a weight of evidence approach, we delimited the Chr 15 QTL to an ∼2 Mb region containing 21 genes (10 protein coding) and nominated three candidate genes, namely Oxr1, Rspo2, and Angpt1. Gene and protein expression data further supported Oxr1 and Angpt1 as priority candidate genes. In summary, we have shown that O3-induced lung injury is modulated by genetic variation, identified two high priority candidate genes, and demonstrated the value of the CC for detecting GxE.

Modeling human heterogeneity of obesity with diversity outbred mice reveals a fat mass-dependent therapeutic window for resolvin E1.

Resolvin E1 (RvE1), a specialized pro-resolving mediator (SPM), improves glucose homeostasis in inbred mouse models of obesity. However, an impediment toward translation is that obesity is a highly heterogenous disease in which individuals will respond very differently to interventions such as RvE1. Thus, there is a need to study SPMs in the context of modeling the heterogeneity of obesity that is observed in humans. We investigated how RvE1 controls the concentration of key circulating metabolic biomarkers using diversity outbred (DO) mice, which mimic human heterogeneity. We first demonstrate that weights of DO mice can be classified into distinct distributions of fat mass (i.e., modeling differing classes of obesity) in response to a high-fat diet and in the human population when examining body composition. Next, we show RvE1 administration based on body weight for four consecutive days after giving mice a high-fat diet led to approximately half of the mice responding positively for serum total gastric inhibitory polypeptide (GIP), glucagon, insulin, glucose, leptin, and resistin. Interestingly, RvE1 improved hyperleptinemia most effectively in the lowest class of fat mass despite adjusting the dose of RvE1 with increasing adiposity. Furthermore, leptin levels after RvE1 treatment were the lowest in those mice that were also RvE1 positive responders for insulin and resistin. Collectively, these results suggest a therapeutic fat mass-dependent window for RvE1, which should be considered in future clinical trials. Moreover, the data underscore the importance of studying SPMs with heterogenous mice as a step toward precision SPM administration in humans.

A diversity outbred F1 mouse model identifies host-intrinsic genetic regulators of response to immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICI) have improved outcomes for a variety of malignancies; however, many patients fail to benefit. While tumor-intrinsic mechanisms are likely involved in therapy resistance, it is unclear to what extent host genetic background influences response. To investigate this, we utilized the Diversity Outbred (DO) and Collaborative Cross (CC) mouse models. DO mice are an outbred stock generated by crossbreeding eight inbred founder strains, and CC mice are recombinant inbred mice generated from the same eight founders. We generated 207 DOB6F1 mice representing 48 DO dams and demonstrated that these mice reliably accept the C57BL/6-syngeneic B16F0 tumor and that host genetic background influences response to ICI. Genetic linkage analysis from 142 mice identified multiple regions including one within chromosome 13 that associated with therapeutic response. We utilized 6 CC strains bearing the positive (NZO) or negative (C57BL/6) driver genotype in this locus. We found that 2/3 of predicted responder CCB6F1 crosses show reproducible ICI response. The chromosome 13 locus contains the murine prolactin family, which is a known immunomodulating cytokine associated with various autoimmune disorders. To directly test whether prolactin influences ICI response rates, we implanted inbred C57BL/6 mice with subcutaneous slow-release prolactin pellets to induce mild hyperprolactinemia. Prolactin augmented ICI response against B16F0, with increased CD8 infiltration and 5/8 mice exhibiting slowed tumor growth relative to controls. This study highlights the role of host genetics in ICI response and supports the use of F1 crosses in the DO and CC mouse populations as powerful cancer immunotherapy models.

Lung metabolome of 1,3-butadiene exposed Collaborative Cross mice reflects metabolic phenotype of human lung cancer.

1,3-Butadiene (BD) exposure is known to cause numerous adverse health effects, including cancer, in animals and humans. BD is metabolized to reactive epoxide intermediates, which are genotoxic, but it is not well know what other effects BD has on cellular metabolism. We examined the effects of exposure to BD on the mouse lung metabolome in the genetically heterogeneous collaborative cross outbred mouse model. Mice were exposed to 3 concentra-tions of BD for 10 days (2, 20, and 200 ppm), and lung tissues were analyzed using high-resolution mass spectrometry-based metabolomics. As compared to controls (0 ppm BD), BD had extensive effects on lung metabolism at all concentrations of exposure, including the lowest concentration of 2 ppm, as reflected by reprogramming of multiple metabolic pathways. Metabolites participating in glycolysis and the tricarboxylic acid cycle were elevated, with 8 out of 10 metabolites demonstrating a 2 to 8-fold increase, including the oncometabolite fumarate. Fatty acid levels, sphingosine, and sphinganine were decreased (2 to 8-fold), and fatty acyl-CoAs were significantly increased (16 to 31-fold), suggesting adjustments in lipid metabolism. Furthermore, metabolites involved in basic amino acid metabolism, steroid hormone metabolism, and nucleic acid metabolism were significantly altered. Overall, these changes mirror the metabolic alterations found in lung cancer cells, suggesting that very low doses of BD induce metabolic adaptations that may prevent or promote adverse health effects such as tumor formation.

Quantitative trait mapping in Diversity Outbred mice identifies novel genomic regions associated with the hepatic glutathione redox system.

The tripeptide glutathione (GSH) is instrumental to antioxidant protection and xenobiotic metabolism, and the ratio of its reduced and oxidized forms (GSH/GSSG) indicates the cellular redox environment and maintains key aspects of cellular signaling. Disruptions in GSH levels and GSH/GSSG have long been tied to various chronic diseases, and many studies have examined whether variant alleles in genes responsible for GSH synthesis and metabolism are associated with increased disease risk. However, past studies have been limited to established, canonical GSH genes, though emerging evidence suggests that novel loci and genes influence the GSH redox system in specific tissues. The present study marks the most comprehensive effort to date to directly identify genetic loci associated with the GSH redox system. We employed the Diversity Outbred (DO) mouse population, a model of human genetics, and measured GSH and the essential redox cofactor NADPH in liver, the organ with the highest levels of GSH in the body. Under normal physiological conditions, we observed substantial variation in hepatic GSH and NADPH levels and their redox balances, and discovered a novel, significant quantitative trait locus (QTL) on murine chromosome 16 underlying GSH/GSSG; bioinformatics analyses revealed Socs1 to be the most likely candidate gene. We also discovered novel QTL associated with hepatic NADP+ levels and NADP+/NADPH, as well as unique candidate genes behind each trait. Overall, these findings transform our understanding of the GSH redox system, revealing genetic loci that govern it and proposing new candidate genes to investigate in future mechanistic endeavors.

Genetic mapping of novel modifiers for ApcMin induced intestinal polyps' development using the genetic architecture power of the collaborative cross mice.

BACKGROUND: Familial adenomatous polyposis is an inherited genetic disease, characterized by colorectal polyps. It is caused by inactivating mutations in the Adenomatous polyposis coli (Apc) gene. Mice carrying a nonsense mutation in the Apc gene at R850, which is designated ApcMin/+ (Multiple intestinal neoplasia), develop intestinal adenomas. Several genetic modifier loci of Min (Mom) were previously mapped, but so far, most of the underlying genes have not been identified. To identify novel modifier loci associated with ApcMin/+, we performed quantitative trait loci (QTL) analysis for polyp development using 49 F1 crosses between different Collaborative Cross (CC) lines and C57BL/6 J-ApcMin/+mice. The CC population is a genetic reference panel of recombinant inbred lines, each line independently descended from eight genetically diverse founder strains. C57BL/6 J-ApcMin/+ males were mated with females from 49 CC lines. F1 offspring were terminated at 23 weeks and polyp counts from three sub-regions (SB1-3) of small intestinal and colon were recorded. RESULTS: The number of polyps in all these sub-regions and colon varied significantly between the different CC lines. At 95% genome-wide significance, we mapped nine novel QTL for variation in polyp number, with distinct QTL associated with each intestinal sub-region. QTL confidence intervals varied in width between 2.63-17.79 Mb. We extracted all genes in the mapped QTL at 90 and 95% CI levels using the BioInfoMiner online platform to extract, significantly enriched pathways and key linker genes, that act as regulatory and orchestrators of the phenotypic landscape associated with the ApcMin/+ mutation. CONCLUSIONS: Genomic structure of the CC lines has allowed us to identify novel modifiers and confirmed some of the previously mapped modifiers. Key genes involved mainly in metabolic and immunological processes were identified. Future steps in this analysis will be to identify regulatory elements - and possible epistatic effects - located in the mapped QTL.

Genetic background influences the effect of thirdhand smoke exposure on anxiety and memory in Collaborative Cross mice.

Growing evidence indicates that thirdhand smoke (THS) exposure induces many adverse health effects. However, it is unclear how THS exposure affects behavior and how host genetic background modulates phenotypic changes. Here we used the Collaborative Cross (CC) mouse population-based model to assess behavioral alterations immediately after THS exposure from 4 to 9 weeks of age. We first measured anxiety-like behavior in six strains using light/dark box combined with a custom multivariate mouse tracking system. We developed an anxiety risk scoring system based on anxiety-related traits and then evaluated the THS impact on them. THS exposure significantly decreased anxiety risk in CC019 (P = 0.002) and CC051 (P = 0.009), but increased anxiety risk in CC036 (P < 0.001), while the other three strains did not show significant changes in anxiety-related traits. Such differences were driven by female mice for the six measures of anxiety-like behavior. Memory potential was measured in the same cohort of mice using the passive avoidance assay. Both THS-exposed male and female CC019 mice displayed significant memory loss compared to controls while no significant changes were found in the other five strains. This study provides strong evidence that THS exposure leads to strain-dependent changes in anxiety-like behavior and memory, suggesting that host genetic variations play a critical role in individual susceptibility to THS-induced effects.

Systems genetics in diversity outbred mice inform BMD GWAS and identify determinants of bone strength.

Genome-wide association studies (GWASs) for osteoporotic traits have identified over 1000 associations; however, their impact has been limited by the difficulties of causal gene identification and a strict focus on bone mineral density (BMD). Here, we use Diversity Outbred (DO) mice to directly address these limitations by performing a systems genetics analysis of 55 complex skeletal phenotypes. We apply a network approach to cortical bone RNA-seq data to discover 66 genes likely to be causal for human BMD GWAS associations, including the genes SERTAD4 and GLT8D2. We also perform GWAS in the DO for a wide-range of bone traits and identify Qsox1 as a gene influencing cortical bone accrual and bone strength. In this work, we advance our understanding of the genetics of osteoporosis and highlight the ability of the mouse to inform human genetics.

Studying the pharmacogenomic effect of cranberry extract on reducing body weight using collaborative cross mice.

The non-dialyzable material (NDM) of polyphenol-rich cranberry extract (CRE) powder (NDM-CRE) was studied for its effect of inducing body weight (BW) loss in 13 different mouse lines with well-defined genetically diverse backgrounds, named the collaborative cross (CC). From the age of 8 weeks, the mice were maintained on a high-fat diet (HFD) for 18 weeks, to induce obesity, and BW was measured biweekly. From week 12, CRE was injected intraperitoneally (IP) (50 mg kg-1) 3 times a week per mouse for a 6 week period. Statistical analysis results have shown a significant increase in body weight between week 0 and week 12; the increase in BW of 13 lines of mice on HFD was in the range of 10.41% to 68.65% for males and 9.78% to 64.74% for females. After injecting NDM-CRE extract, our analysis has shown an induced change in BW between week 12 and week 18. In males, NDM-CRE caused a significant decrease in BW of 5 out of the 13 lines in the range of -5.68% to -16.69% and a significant increase of 8.31% in BW of one male line, whereas in seven lines there was no significant decrease (-2.14% to -4.09%). In females, NDM-CRE caused a significant decrease in BW of 5 out of the 13 lines in the range of -3.90% to -11.83%, whereas in eight lines there were no significant changes in BW and it ranged between -1.50% and 4.90%. The broad-sense heritability (H2) and genetic coefficient of variation (CVg) were estimated and found to be between 0.71 and 0.81 for H2, and 0.18 and 0.24 for CVg of females and males, respectively, with respect to the efficacy of NDM-CRE on body weight reduction. Our results have shown that hosts with different genetic backgrounds respond differently to body weight increase, as well as to NDM-CRE treatment for body weight reduction. These results provide a platform for assessing more CC lines and mapping genes underlying the efficacy of the NDM-CRE treatment as a way of understanding pharmacogenomics.

Studying host genetic background effects on multimorbidity of intestinal cancer development, type 2 diabetes and obesity in response to oral bacterial infection and high-fat diet using the collaborative cross (CC) lines.

Background: Multimorbidity of intestinal cancer (IC), type 2 diabetes (T2D) and obesity is a complex set of diseases, affected by environmental and genetic risk factors. High-fat diet (HFD) and oral bacterial infection play important roles in the etiology of these diseases through inflammation and various biological mechanisms. Methods: To study the complexity of this multimorbidity, we used the collaborative cross (CC) mouse genetics reference population. We aimed to study the multimorbidity of IC, T2D, and obesity using CC lines, measuring their responses to HFD and oral bacterial infection. The study used 63 mice of both sexes generated from two CC lines (IL557 and IL711). For 12 weeks, experimental mice were maintained on specific dietary regimes combined with co-infection with oral bacteria Porphyromonas gingivalis and Fusobacterium nucleatum, while control groups were not infected. Body weight (BW) and results of a intraperitoneal glucose tolerance test (IPGTT) were recorded at the end of 12 weeks, after which length and size of the intestines were assessed for polyp counts. Results: Polyp counts ranged between 2 and 10 per CC line. The combination of HFD and infection significantly reduced (P < .01) the colon polyp size of IL557 females to 2.5 cm2, compared to the other groups. Comparing BW gain, IL557 males on HFD gained 18 g, while the females gained 10 g under the same conditions and showed the highest area under curve (AUC) values of 40 000-45 000 (min mg/dL) in the IPGTT. Conclusion: The results show that mice from different genetic backgrounds respond differently to a high fat diet and oral infection in terms of polyp development and glucose tolerance, and this effect is gender related.

Host genetics and gut microbiota cooperatively contribute to azoxymethane-induced acute toxicity in Collaborative Cross mice.

Azoxymethane (AOM) is a widely used carcinogen to study chemical-induced colorectal carcinogenesis and is an agent for studying fulminant hepatic failure. The inter-strain susceptibility to acute toxicity by AOM has been reported, but its association with host genetics or gut microbiota remains largely unexplored. Here a cohort of genetically diverse Collaborative Cross (CC) mice was used to assess the contribution of host genetics and the gut microbiome to AOM-induced acute toxicity. We observed variation in AOM-induced acute liver failure across CC strains. Quantitative trait loci (QTL) analysis revealed three chromosome regions significantly associated with AOM toxicity. Genes located within these QTL, including peroxisome proliferator-activated receptor alpha (Ppara), were enriched for enzyme activator and nucleoside-triphosphatase regulator activity. We further demonstrated that the protein level of PPARα in liver tissues from sensitive strains was remarkably lower compared to levels in resistant strains, consistent with protective role of PPAR family in liver injury. We discovered that the abundance levels of gut microbial families Anaeroplasmataceae, Ruminococcaceae, Lactobacillaceae, Akkermansiaceae and Clostridiaceae were significantly higher in the sensitive strains compared to the resistant strains. Using a random forest classifier method, we determined that the relative abundance levels of these microbial families predicted AOM toxicity with the area under the receiver-operating curve (AUC) of 0.75. Combining the three genetic loci and five microbial families increased the predictive accuracy of AOM toxicity (AUC of 0.99). Moreover, we found that Ruminococcaceae and Lactobacillaceae acted as mediators between host genetics and AOM toxicity. In conclusion, this study shows that host genetics and specific microbiome members play a critical role in AOM-induced acute toxicity, which provides a framework for analysis of the health effects from environmental toxicants.

Development of a Model of the Acute and Delayed Effects of High Dose Radiation Exposure in Jackson Diversity Outbred Mice; Comparison to Inbred C57BL/6 Mice.

Development of medical countermeasures against radiation relies on robust animal models for efficacy testing. Mouse models have advantages over larger species due to economics, ease of conducting aging studies, existence of historical databases, and research tools allowing for sophisticated mechanistic studies. However, the radiation dose-response relationship of inbred strains is inherently steep and sensitive to experimental variables, and inbred models have been criticized for lacking genetic diversity. Jackson Diversity Outbred (JDO) mice are the most genetically diverse strain available, developed by the Collaborative Cross Consortium using eight founder strains, and may represent a more accurate model of humans than inbred strains. Herein, models of the Hematopoietic-Acute Radiation Syndrome and the Delayed Effects of Acute Radiation Exposure were developed in JDO mice and compared to inbred C57BL/6. The dose response relationship curve in JDO mice mirrored the more shallow curves of primates and humans, characteristic of genetic diversity. JDO mice were more radioresistant than C57BL/6 and differed in sensitivity to antibiotic countermeasures. The model was validated with pegylated-G-CSF, which provided significantly enhanced 30-d survival and accelerated blood recovery. Long-term JDO survivors exhibited increased recovery of blood cells and functional bone marrow hematopoietic progenitors compared to C57BL/6. While JDO hematopoietic stem cells declined more in number, they maintained a greater degree of quiescence compared to C57BL/6, which is essential for maintaining function. These JDO radiation models offer many of the advantages of small animals with the genetic diversity of large animals, providing an attractive alternative to currently available radiation animal models.