Mapping of susceptibility loci for Ebola virus pathogenesis in mice.

Ebola virus (EBOV), a major global health concern, causes severe, often fatal EBOV disease (EVD) in humans. Host genetic variation plays a critical role, yet the identity of host susceptibility loci in mammals remains unknown. Using genetic reference populations, we generate an F2 mapping cohort to identify host susceptibility loci that regulate EVD. While disease-resistant mice display minimal pathogenesis, susceptible mice display severe liver pathology consistent with EVD-like disease and transcriptional signatures associated with inflammatory and liver metabolic processes. A significant quantitative trait locus (QTL) for virus RNA load in blood is identified in chromosome (chr)8, and a severe clinical disease and mortality QTL is mapped to chr7, which includes the Trim5 locus. Using knockout mice, we validate the Trim5 locus as one potential driver of liver failure and mortality after infection. The identification of susceptibility loci provides insight into molecular genetic mechanisms regulating EVD progression and severity, potentially informing therapeutics and vaccination strategies.

A mutation in Themis contributes to anaphylaxis severity following oral peanut challenge in CC027 mice.

BACKGROUND: The development of peanut allergy is due to a combination of genetic and environmental factors, although specific genes have proven difficult to identify. Previously, we reported that peanut-sensitized CC027/GeniUnc (CC027) mice develop anaphylaxis upon oral challenge to peanut, unlike C3H/HeJ (C3H) mice. OBJECTIVE: To determine the genetic basis of orally-induced anaphylaxis to peanut in CC027 mice. METHODS: A genetic mapping population between CC027 and C3H mice was designed to identify the genetic factors that drive oral anaphylaxis. A total of 356 CC027xC3H backcrossed mice were generated, sensitized to peanut, then challenged to peanut by oral gavage. Anaphylaxis and peanut-specific IgE were quantified for all mice. T-cell phenotyping was conducted on CC027 and five additional CC strains. RESULTS: Anaphylaxis to peanut was absent in 77% of backcrossed mice, with 19% showing moderate anaphylaxis, and 4% having severe anaphylaxis. A total of eight genetic loci were associated with variation in response to peanut challenge, six associated with anaphylaxis (temperature decrease) and two associated with peanut-specific IgE levels. There were two major loci that impacted multiple aspects of the severity of acute anaphylaxis, at which the CC027 allele was associated with worse outcome. At one of these loci, CC027 has a private genetic variant in the Themis (thymocyte-expressed molecule involved in selection) gene. Consistent with Themis' described functions, we found that CC027 have more immature T cells with fewer CD8+, CD4+, and CD4+CD25+CD127- regulatory T cells. CONCLUSION: Our results demonstrate a key role for Themis in the orally-reactive CC027 mouse model of peanut allergy.

Genetic loci regulate Sarbecovirus pathogenesis: A comparison across mice and humans.

Coronavirus (CoV) cause considerable morbidity and mortality in humans and other mammals, as evidenced by the emergence of Severe Acute Respiratory CoV (SARS-CoV) in 2003, Middle East Respiratory CoV (MERS-CoV) in 2012, and SARS-CoV-2 in 2019. Although poorly characterized, natural genetic variation in human and other mammals modulate virus pathogenesis, as reflected by the spectrum of clinical outcomes ranging from asymptomatic infections to lethal disease. Using multiple human epidemic and zoonotic Sarbecoviruses, coupled with murine Collaborative Cross genetic reference populations, we identify several dozen quantitative trait loci that regulate SARS-like group-2B CoV pathogenesis and replication. Under a Chr4 QTL, we deleted a candidate interferon stimulated gene, Trim14 which resulted in enhanced SARS-CoV titers and clinical disease, suggesting an antiviral role during infection. Importantly, about 60 % of the murine QTL encode susceptibility genes identified as priority candidates from human genome-wide association studies (GWAS) studies after SARS-CoV-2 infection, suggesting that similar selective forces have targeted analogous genes and pathways to regulate Sarbecovirus disease across diverse mammalian hosts. These studies provide an experimental platform in rodents to investigate the molecular-genetic mechanisms by which potential cross mammalian susceptibility loci and genes regulate type-specific and cross-SARS-like group 2B CoV replication, immunity, and pathogenesis in rodent models. Our study also provides a paradigm for identifying susceptibility loci for other highly heterogeneous and virulent viruses that sporadically emerge from zoonotic reservoirs to plague human and animal populations.

Unique immune profiles in collaborative cross mice linked to survival and viral clearance upon infection.

The response to infection is generally heterogeneous and diverse, with some individuals remaining asymptomatic while others present with severe disease or a diverse range of symptoms. Here, we address the role of host genetics on immune phenotypes and clinical outcomes following viral infection by studying genetically diverse mice from the Collaborative Cross (CC), allowing for use of a small animal model with controlled genetic diversity while maintaining genetic replicates. We demonstrate variation by deeply profiling a broad range of innate and adaptive immune cell phenotypes at steady-state in 63 genetically distinct CC mouse strains and link baseline immune signatures with virologic and clinical disease outcomes following infection of mice with herpes simplex virus 2 (HSV-2) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This work serves as a resource for CC strain selection based on steady-state immune phenotypes or disease presentation upon viral infection, and further, points to possible pre-infection immune correlates of survival and early viral clearance upon infection.

Host Genetic Variation Impacts SARS-CoV-2 Vaccination Response in the Diversity Outbred Mouse Population.

The COVID-19 pandemic led to the rapid and worldwide development of highly effective vaccines against SARS-CoV-2. However, there is significant individual-to-individual variation in vaccine efficacy due to factors including viral variants, host age, immune status, environmental and host genetic factors. Understanding those determinants driving this variation may inform the development of more broadly protective vaccine strategies. While host genetic factors are known to impact vaccine efficacy for respiratory pathogens such as influenza and tuberculosis, the impact of host genetic variation on vaccine efficacy against COVID-19 is not well understood. To model the impact of host genetic variation on SARS-CoV-2 vaccine efficacy, while controlling for the impact of non-genetic factors, we used the Diversity Outbred (DO) mouse model. We found that DO mice immunized against SARS-CoV-2 exhibited high levels of variation in vaccine-induced neutralizing antibody responses. While the majority of the vaccinated mice were protected from virus-induced disease, similar to human populations, we observed vaccine breakthrough in a subset of mice. Importantly, we found that this variation in neutralizing antibody, virus-induced disease, and viral titer is heritable, indicating that the DO serves as a useful model system for studying the contribution of genetic variation of both vaccines and disease outcomes.

A mutation in Themis contributes to peanut-induced oral anaphylaxis in CC027 mice.

Background: The development of peanut allergy is due to a combination of genetic and environmental factors, although specific genes have proven difficult to identify. Previously, we reported that peanut-sensitized CC027/GeniUnc (CC027) mice develop anaphylaxis upon oral challenge to peanut, unlike C3H/HeJ (C3H) mice. Objective: To determine the genetic basis of orally-induced anaphylaxis to peanut in CC027 mice. Methods: A genetic mapping population between CC027 and C3H mice was designed to identify the genetic factors that drive oral anaphylaxis. A total of 356 CC027xC3H backcrossed mice were generated, sensitized to peanut, then challenged to peanut by oral gavage. Anaphylaxis and peanut-specific IgE were quantified for all mice. T-cell phenotyping was conducted on CC027 and five additional CC strains. Results: Anaphylaxis to peanut was absent in 77% of backcrossed mice, with 19% showing moderate anaphylaxis, and 4% having severe anaphylaxis. A total of eight genetic loci were associated with variation in response to peanut challenge, six associated with anaphylaxis (temperature decrease) and two associated with peanut-specific IgE levels. There were two major loci that impacted multiple aspects of the severity of acute anaphylaxis, at which the CC027 allele was associated with worse outcome. At one of these loci, CC027 has a private genetic variant in the Themis (thymocyte-expressed molecule involved in selection) gene. Consistent with Themis' described functions, we found that CC027 have more immature T cells with fewer CD8+, CD4+, and CD4+CD25+CD127- regulatory T cells. Conclusion: Our results demonstrate a key role for Themis in the orally-reactive CC027 mouse model of peanut allergy.

Large differences in collateral blood vessel abundance among individuals arise from multiple genetic variants.

Collateral blood flow varies greatly among humans for reasons that remain unclear, resulting in significant differences in ischemic tissue damage. A similarly large variation has also been found in mice that is caused by genetic background-dependent differences in the extent of collateral formation, termed collaterogenesis-a unique angiogenic process that occurs during development and determines collateral number and diameter in the adult. Previous studies have identified several quantitative trait loci (QTL) linked to this variation. However, understanding has been hampered by the use of closely related inbred strains that do not model the wide genetic variation present in the "outbred" human population. The Collaborative Cross (CC) multiparent mouse genetic reference panel was developed to address this limitation. Herein we measured the number and average diameter of cerebral collaterals in 60 CC strains, their 8 founder strains, 8 F1 crosses of CC strains selected for abundant versus sparse collaterals, and 2 intercross populations created from the latter. Collateral number evidenced 47-fold variation among the 60 CC strains, with 14% having poor, 25% poor-to-intermediate, 47% intermediate-to-good, and 13% good collateral abundance, that was associated with large differences in post-stroke infarct volume. Collateral number in skeletal muscle and intestine of selected high- and low-collateral strains evidenced the same relative abundance as in brain. Genome-wide mapping demonstrated that collateral abundance is a highly polymorphic trait. Subsequent analysis identified: 6 novel QTL circumscribing 28 high-priority candidate genes harboring putative loss-of-function polymorphisms (SNPs) associated with low collateral number; 335 predicted-deleterious SNPs present in their human orthologs; and 32 genes associated with vascular development but lacking protein coding variants. Six additional suggestive QTL (LOD > 4.5) were also identified in CC-wide QTL mapping. This study provides a comprehensive set of candidate genes for future investigations aimed at identifying signaling proteins within the collaterogenesis pathway whose variants potentially underlie genetic-dependent collateral insufficiency in brain and other tissues.

Large differences in collateral blood vessel abundance among individuals arise from multiple genetic variants.

Collateral blood flow varies greatly among humans for reasons that remain unclear, resulting in significant differences in ischemic tissue damage. A similarly large variation has also been found in mice that is caused by genetic background-dependent differences in the extent of collateral formation, termed collaterogenesis-a unique angiogenic process that occurs during development and determines collateral number and diameter in the adult. Previous studies have identified several quantitative trait loci (QTL) linked to this variation. However, understanding has been hampered by the use of closely related inbred strains that do not model the wide genetic variation present in the "outbred" human population. The Collaborative Cross (CC) multiparent mouse genetic reference panel was developed to address this limitation. Herein we measured the number and average diameter of cerebral collaterals in 60 CC strains, their 8 founder strains, 8 F1 crosses of CC strains selected for abundant versus sparse collaterals, and 2 intercross populations created from the latter. Collateral number evidenced 47-fold variation among the 60 CC strains, with 14% having poor, 25% poor-to-intermediate, 47% intermediate-to-good, and 13% good collateral abundance, that was associated with large differences in post-stroke infarct volume. Genome-wide mapping demonstrated that collateral abundance is a highly polymorphic trait. Subsequent analysis identified: 6 novel QTL circumscribing 28 high-priority candidate genes harboring putative loss-of-function polymorphisms (SNPs) associated with low collateral number; 335 predicted-deleterious SNPs present in their human orthologs; and 32 genes associated with vascular development but lacking protein coding variants. This study provides a comprehensive set of candidate genes for future investigations aimed at identifying signaling proteins within the collaterogenesis pathway whose variants potentially underlie genetic-dependent collateral insufficiency in brain and other tissues.

Memory T cells possess an innate-like function in local protection from mucosal infection.

Mucosal infections pose a significant global health burden. Antigen-specific tissue-resident T cells are critical to maintaining barrier immunity. Previous studies in the context of systemic infection suggest that memory CD8+ T cells may also provide innate-like protection against antigenically unrelated pathogens independent of T cell receptor engagement. Whether bystander T cell activation is also an important defense mechanism in the mucosa is poorly understood. Here, we investigated whether innate-like memory CD8+ T cells could protect against a model mucosal virus infection, herpes simplex virus 2 (HSV-2). We found that immunization with an irrelevant antigen delayed disease progression from lethal HSV-2 challenge, suggesting that memory CD8+ T cells may mediate protection despite the lack of antigen specificity. Upon HSV-2 infection, we observed an early infiltration, rather than substantial local proliferation, of antigen-nonspecific CD8+ T cells, which became bystander-activated only within the infected mucosal tissue. Critically, we show that bystander-activated CD8+ T cells are sufficient to reduce early viral burden after HSV-2 infection. Finally, local cytokine cues within the tissue microenvironment after infection were sufficient for bystander activation of mucosal tissue memory CD8+ T cells from mice and humans. Altogether, our findings suggest that local bystander activation of CD8+ memory T cells contributes a fast and effective innate-like response to infection in mucosal tissue.

Multi-omics analysis identifies drivers of protein phosphorylation.

BACKGROUND: Phosphorylation of proteins is a key step in the regulation of many cellular processes including activation of enzymes and signaling cascades. The abundance of a phosphorylated peptide (phosphopeptide) is determined by the abundance of its parent protein and the proportion of target sites that are phosphorylated. RESULTS: We quantified phosphopeptides, proteins, and transcripts in heart, liver, and kidney tissue samples of mice from 58 strains of the Collaborative Cross strain panel. We mapped ~700 phosphorylation quantitative trait loci (phQTL) across the three tissues and applied genetic mediation analysis to identify causal drivers of phosphorylation. We identified kinases, phosphatases, cytokines, and other factors, including both known and potentially novel interactions between target proteins and genes that regulate site-specific phosphorylation. Our analysis highlights multiple targets of pyruvate dehydrogenase kinase 1 (PDK1), a regulator of mitochondrial function that shows reduced activity in the NZO/HILtJ mouse, a polygenic model of obesity and type 2 diabetes. CONCLUSIONS: Together, this integrative multi-omics analysis in genetically diverse CC strains provides a powerful tool to identify regulators of protein phosphorylation. The data generated in this study provides a resource for further exploration.

Forward genetic screen of homeostatic antibody levels in the Collaborative Cross identifies MBD1 as a novel regulator of B cell homeostasis.

Variation in immune homeostasis, the state in which the immune system is maintained in the absence of stimulation, is highly variable across populations. This variation is attributed to both genetic and environmental factors. However, the identity and function of specific regulators have been difficult to identify in humans. We evaluated homeostatic antibody levels in the serum of the Collaborative Cross (CC) mouse genetic reference population. We found heritable variation in all antibody isotypes and subtypes measured. We identified 4 quantitative trait loci (QTL) associated with 3 IgG subtypes: IgG1, IgG2b, and IgG2c. While 3 of these QTL map to genome regions of known immunological significance (major histocompatibility and immunoglobulin heavy chain locus), Qih1 (associated with variation in IgG1) mapped to a novel locus on Chromosome 18. We further associated this locus with B cell proportions in the spleen and identify Methyl-CpG binding domain protein 1 under this locus as a novel regulator of homeostatic IgG1 levels in the serum and marginal zone B cells (MZB) in the spleen, consistent with a role in MZB differentiation to antibody secreting cells.

An interaction of inorganic arsenic exposure with body weight and composition on type 2 diabetes indicators in Diversity Outbred mice.

Type 2 diabetes (T2D) is a complex metabolic disorder with no cure and high morbidity. Exposure to inorganic arsenic (iAs), a ubiquitous environmental contaminant, is associated with increased T2D risk. Despite growing evidence linking iAs exposure to T2D, the factors underlying inter-individual differences in susceptibility remain unclear. This study examined the interaction between chronic iAs exposure and body composition in a cohort of 75 Diversity Outbred mice. The study design mimics that of an exposed human population where the genetic diversity of the mice provides the variation in response, in contrast to a design that includes untreated mice. Male mice were exposed to iAs in drinking water (100 ppb) for 26 weeks. Metabolic indicators used as diabetes surrogates included fasting blood glucose and plasma insulin (FBG, FPI), blood glucose and plasma insulin 15 min after glucose challenge (BG15, PI15), homeostatic model assessment for [Formula: see text]-cell function and insulin resistance (HOMA-B, HOMA-IR), and insulinogenic index. Body composition was determined using magnetic resonance imaging, and the concentrations of iAs and its methylated metabolites were measured in liver and urine. Associations between cumulative iAs consumption and FPI, PI15, HOMA-B, and HOMA-IR manifested as significant interactions between iAs and body weight/composition. Arsenic speciation analyses in liver and urine suggest little variation in the mice's ability to metabolize iAs. The observed interactions accord with current research aiming to disentangle the effects of multiple complex factors on T2D risk, highlighting the need for further research on iAs metabolism and its consequences in genetically diverse mouse strains.

A Multitrait Locus Regulates Sarbecovirus Pathogenesis.

Infectious diseases have shaped the human population genetic structure, and genetic variation influences the susceptibility to many viral diseases. However, a variety of challenges have made the implementation of traditional human Genome-wide Association Studies (GWAS) approaches to study these infectious outcomes challenging. In contrast, mouse models of infectious diseases provide an experimental control and precision, which facilitates analyses and mechanistic studies of the role of genetic variation on infection. Here we use a genetic mapping cross between two distinct Collaborative Cross mouse strains with respect to severe acute respiratory syndrome coronavirus (SARS-CoV) disease outcomes. We find several loci control differential disease outcome for a variety of traits in the context of SARS-CoV infection. Importantly, we identify a locus on mouse chromosome 9 that shows conserved synteny with a human GWAS locus for SARS-CoV-2 severe disease. We follow-up and confirm a role for this locus, and identify two candidate genes, CCR9 and CXCR6, that both play a key role in regulating the severity of SARS-CoV, SARS-CoV-2, and a distantly related bat sarbecovirus disease outcomes. As such we provide a template for using experimental mouse crosses to identify and characterize multitrait loci that regulate pathogenic infectious outcomes across species. IMPORTANCE Host genetic variation is an important determinant that predicts disease outcomes following infection. In the setting of highly pathogenic coronavirus infections genetic determinants underlying host susceptibility and mortality remain unclear. To elucidate the role of host genetic variation on sarbecovirus pathogenesis and disease outcomes, we utilized the Collaborative Cross (CC) mouse genetic reference population as a model to identify susceptibility alleles to SARS-CoV and SARS-CoV-2 infections. Our findings reveal that a multitrait loci found in chromosome 9 is an important regulator of sarbecovirus pathogenesis in mice. Within this locus, we identified and validated CCR9 and CXCR6 as important regulators of host disease outcomes. Specifically, both CCR9 and CXCR6 are protective against severe SARS-CoV, SARS-CoV-2, and SARS-related HKU3 virus disease in mice. This chromosome 9 multitrait locus may be important to help identify genes that regulate coronavirus disease outcomes in humans.

A Multitrait Locus Regulates Sarbecovirus Pathogenesis.

Infectious diseases have shaped the human population genetic structure, and genetic variation influences the susceptibility to many viral diseases. However, a variety of challenges have made the implementation of traditional human Genome-wide Association Studies (GWAS) approaches to study these infectious outcomes challenging. In contrast, mouse models of infectious diseases provide an experimental control and precision, which facilitates analyses and mechanistic studies of the role of genetic variation on infection. Here we use a genetic mapping cross between two distinct Collaborative Cross mouse strains with respect to SARS-CoV disease outcomes. We find several loci control differential disease outcome for a variety of traits in the context of SARS-CoV infection. Importantly, we identify a locus on mouse Chromosome 9 that shows conserved synteny with a human GWAS locus for SARS-CoV-2 severe disease. We follow-up and confirm a role for this locus, and identify two candidate genes, CCR9 and CXCR6 that both play a key role in regulating the severity of SARS-CoV, SARS-CoV-2 and a distantly related bat sarbecovirus disease outcomes. As such we provide a template for using experimental mouse crosses to identify and characterize multitrait loci that regulate pathogenic infectious outcomes across species.

Host-pathogen genetic interactions underlie tuberculosis susceptibility in genetically diverse mice.

The outcome of an encounter with Mycobacterium tuberculosis (Mtb) depends on the pathogen's ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to create a resource for associating bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb transposon mutant fitness (TnSeq) across the CC panel revealed that many virulence pathways are only required in specific host microenvironments, identifying a large fraction of the pathogen's genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits can be associated with genetic variants distributed across the mouse genome, making the CC a unique population for identifying specific host-pathogen genetic interactions that influence pathogenesis.

Common Mechanism of SARS-CoV and SARS-CoV-2 Pathogenesis across Species.

Sarbecovirus (CoV) infections, including Severe Acute Respiratory CoV (SARS-CoV) and SARS-CoV-2, are considerable human threats. Human GWAS studies have recently identified loci associated with variation in SARS-CoV-2 susceptibility. However, genetically tractable models that reproduce human CoV disease outcomes are needed to mechanistically evaluate genetic determinants of CoV susceptibility. We used the Collaborative Cross (CC) and human GWAS datasets to elucidate host susceptibility loci that regulate CoV infections and to identify host quantitative trait loci that modulate severe CoV and pan-CoV disease outcomes including a major disease regulating loci including CCR9. CCR9 ablation resulted in enhanced titer, weight loss, respiratory dysfunction, mortality, and inflammation, providing mechanistic support in mitigating protection from severe SARS-CoV-2 pathogenesis across species. This study represents a comprehensive analysis of susceptibility loci for an entire genus of human pathogens conducted, identifies a large collection of susceptibility loci and candidate genes that regulate multiple aspects type-specific and cross-CoV pathogenesis, and also validates the paradigm of using the CC platform to identify common cross-species susceptibility loci and genes for newly emerging and pre-epidemic viruses.

Regulation of protein abundance in genetically diverse mouse populations.

Genetically diverse mouse populations are powerful tools for characterizing the regulation of the proteome and its relationship to whole-organism phenotypes. We used mass spectrometry to profile and quantify the abundance of 6,798 proteins in liver tissue from mice of both sexes across 58 Collaborative Cross (CC) inbred strains. We previously collected liver proteomics data from the related Diversity Outbred (DO) mice and their founder strains. We show concordance across the proteomics datasets despite being generated from separate experiments, allowing comparative analysis. We map protein abundance quantitative trait loci (pQTLs), identifying 1,087 local and 285 distal in the CC mice and 1,706 local and 414 distal in the DO mice. We find that regulatory effects on individual proteins are conserved across the mouse populations, in particular for local genetic variation and sex differences. In comparison, proteins that form complexes are often co-regulated, displaying varying genetic architectures, and overall show lower heritability and map fewer pQTLs. We have made this resource publicly available to enable quantitative analyses of the regulation of the proteome.

Content and Performance of the MiniMUGA Genotyping Array: A New Tool To Improve Rigor and Reproducibility in Mouse Research.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.

Collaborative Cross mice reveal extreme epilepsy phenotypes and genetic loci for seizure susceptibility.

OBJECTIVE: Animal studies remain essential for understanding mechanisms of epilepsy and identifying new therapeutic targets. However, existing animal models of epilepsy do not reflect the high level of genetic diversity found in the human population. The Collaborative Cross (CC) population is a genetically diverse recombinant inbred panel of mice. The CC offers large genotypic and phenotypic diversity, inbred strains with stable genomes that allow for repeated phenotypic measurements, and genomic tools including whole genome sequence to identify candidate genes and candidate variants. METHODS: We evaluated multiple complex epileptic traits in a sampling of 35 CC inbred strains using the flurothyl-induced seizure and kindling paradigm. We created an F2 population of 297 mice with extreme seizure susceptibility and performed quantitative trait loci (QTL) mapping to identify genomic regions associated with seizure sensitivity. We used quantitative RNA sequencing from CC hippocampal tissue to identify candidate genes and whole genome sequence to identify genetic variants likely affecting gene expression. RESULTS: We identified new mouse models with extreme seizure susceptibility, seizure propagation, epileptogenesis, and SUDEP (sudden unexpected death in epilepsy). We performed QTL mapping and identified one known and seven novel loci associated with seizure sensitivity. We combined whole genome sequencing and hippocampal gene expression to pinpoint biologically plausible candidate genes (eg, Gabra2) and variants associated with seizure sensitivity. SIGNIFICANCE: New mouse models of epilepsy are needed to better understand the complex genetic architecture of seizures and to identify therapeutics. We performed a phenotypic screen utilizing a novel genetic reference population of CC mice. The data we provide enable the identification of protective/risk genes and novel molecular mechanisms linked to complex seizure traits that are currently challenging to study and treat.

Functionally Overlapping Variants Control Tuberculosis Susceptibility in Collaborative Cross Mice.

Host genetics plays an important role in determining the outcome of Mycobacterium tuberculosis infection. We previously found that Collaborative Cross (CC) mouse strains differ in their susceptibility to M. tuberculosis and that the CC042/GeniUnc (CC042) strain suffered from a rapidly progressive disease and failed to produce the protective cytokine gamma interferon (IFN-γ) in the lung. Here, we used parallel genetic and immunological approaches to investigate the basis of CC042 mouse susceptibility. Using a population derived from a CC001/Unc (CC001) × CC042 intercross, we mapped four quantitative trait loci (QTL) underlying tuberculosis immunophenotypes (Tip1 to Tip4). These included QTL that were associated with bacterial burden, IFN-γ production following infection, and an IFN-γ-independent mechanism of bacterial control. Further immunological characterization revealed that CC042 animals recruited relatively few antigen-specific T cells to the lung and that these T cells failed to express the integrin alpha L (αL; i.e., CD11a), which contributes to T cell activation and migration. These defects could be explained by a CC042 private variant in the Itgal gene, which encodes CD11a and is found within the Tip2 interval. This 15-bp deletion leads to aberrant mRNA splicing and is predicted to result in a truncated protein product. The ItgalCC042 genotype was associated with all measured disease traits, indicating that this variant is a major determinant of susceptibility in CC042 mice. The combined effect of functionally distinct Tip variants likely explains the profound susceptibility of CC042 mice and highlights the multigenic nature of tuberculosis control in the Collaborative Cross.IMPORTANCE The variable outcome of Mycobacterium tuberculosis infection observed in natural populations is difficult to model in genetically homogeneous small-animal models. The newly developed Collaborative Cross (CC) represents a reproducible panel of genetically diverse mice that display a broad range of phenotypic responses to infection. We explored the genetic basis of this variation, focusing on a CC line that is highly susceptible to M. tuberculosis infection. This study identified multiple quantitative trait loci associated with bacterial control and cytokine production, including one that is caused by a novel loss-of-function mutation in the Itgal gene, which is necessary for T cell recruitment to the infected lung. These studies verify the multigenic control of mycobacterial disease in the CC panel, identify genetic loci controlling diverse aspects of pathogenesis, and highlight the utility of the CC resource.