Mouse Phenome Project Research Articles

Interfrontal Bone Among Inbred Strains of Mice and QTL Mapping.

The interfrontal bone (IF) is a minor skeletal trait residing between the frontal bones. IF is considered a quasi-continuous trait. Genetic and environmental factors appear to play roles in its development. The mechanism(s) underlying IF bone development are poorly understood. We sought to survey inbred strains of mice for the prevalence of IF and to perform QTL mapping studies. Archived mouse skulls from a mouse phenome project (MPP) were available for this study. 27 inbred strains were investigated with 6–20 mice examined for each strain. Skulls were viewed dorsally and the IF measured using a zoom stereomicroscope equipped with a calibrated reticle. A two generation cross between C3H/HeJ and C57BL/6J mice was performed to generate a panel of 468 F2 mice. F2 mice were phenotyped for presence or absence of IF bone and among mice with the IF bone maximum widths and lengths were measured. F2 mice were genotyped for 573 SNP markers informative between the two strains and subjected to linkage map construction and interval QTL mapping. Results: Strain dependent differences in the prevalence of IF bones were observed. Overall, 77.8% or 21/27, of the inbred strains examined had IF bones. Six strains (C3H/HeJ, MOLF/EiJ, NZW/LacJ, SPRET/EiJ, SWR/J, and WSB/EiJ) lack IF bones. Among the strains with IF bones, the prevalence ranged from 100% for C57BL/6J, C57/LJ, CBA/J, and NZB/B1NJ and down to 5% for strains such as CAST/Ei. QTL mapping for IF bone length and widths identifies for each trait one strong QTL detected on chromosome 14 along with several other significant QTLs on chromosomes 3, 4, 7, and 11. Strain dependent differences in IF will facilitate investigation of genetic factors contributing to IF development. IF bone formation may be a model to understand intrasutural bone formation.

Mouse Phenome Database

The Mouse Phenome Database (MPD; phenome.jax.org) was launched in 2001 as the data coordination center for the international Mouse Phenome Project. MPD integrates quantitative phenotype, gene expression and genotype data into a common annotated framework to facilitate query and analysis. MPD contains >3500 phenotype measurements or traits relevant to human health, including cancer, aging, cardiovascular disorders, obesity, infectious disease susceptibility, blood disorders, neurosensory disorders, drug addiction and toxicity. Since our 2012 NAR report, we have added >70 new data sets, including data from Collaborative Cross lines and Diversity Outbred mice. During this time we have completely revamped our homepage, improved search and navigational aspects of the MPD application, developed several web-enabled data analysis and visualization tools, annotated phenotype data to public ontologies, developed an ontology browser and released new single nucleotide polymorphism query functionality with much higher density coverage than before. Here, we summarize recent data acquisitions and describe our latest improvements.

An Integrative Genomic Analysis of the Superior Fecundity Phenotype in QSi5 Mice

Laboratory inbred mouse models are a valuable resource to identify quantitative trait loci (QTL) for complex reproductive performance traits. Advances in mouse genomics and high density single nucleotide polymorphism mapping has enabled genome-wide association studies to identify genes linked with specific phenotypes. Gene expression profiles of reproductive tissues also provide potentially useful information for identifying genes that play an important role. We have developed a highly fecund inbred strain, QSi5, with accompanying genotyping for comparative analysis of reproductive performance. Here we analyzed the QSi5 phenotype using a comparative analysis with fecundity data derived from 22 inbred strains of mice from the Mouse Phenome Project, and integration with published expression data from mouse ovary development. Using a haplotype association approach, 400 fecundity-associated regions (FDR<0.05) with 499 underlying genes were identified. The most significant associations were located on Chromosomes 14, 8, and 6, and the genes underlying these regions were extracted. When these genes were analyzed for expression in an ovarian development profile (GSE6916) several distinctive co-expression patterns across each developmental stage were identified. The genetic analysis also refined 21 fecundity associated intervals on Chromosomes 1, 6, 9, 13, and 17 that overlapped with previously reported reproductive performance QTL. The combined use of phenotypic and in silico data with an integrative genomic analysis provides a powerful tool for elucidating the molecular mechanisms underlying fecundity.

Mouse Phenome Database (MPD)

The Mouse Phenome Project was launched a decade ago to complement mouse genome sequencing efforts by promoting new phenotyping initiatives under standardized conditions and collecting the data in a central public database, the Mouse Phenome Database (MPD; http://phenome.jax.org). MPD houses a wealth of strain characteristics data to facilitate the use of the laboratory mouse in translational research for human health and disease, helping alleviate problems involving experimentation in humans that cannot be done practically or ethically. Data sets are voluntarily contributed by researchers from a variety of institutions and settings, or in some cases, retrieved by MPD staff from public sources. MPD maintains a growing collection of standardized reference data that assists investigators in selecting mouse strains for research applications; houses treatment/control data for drug studies and other interventions; offers a standardized platform for discovering genotype–phenotype relationships; and provides tools for hypothesis testing. MPD improvements and updates since our last NAR report are presented, including the addition of new tools and features to facilitate navigation and data mining as well as the acquisition of new data (phenotypic, genotypic and gene expression).

Antipsychotic-induced vacuous chewing movements and extrapyramidal side effects are highly heritable in mice

Pharmacogenomics is yet to fulfill its promise of manifestly altering clinical medicine. As one example, a predictive test for tardive dyskinesia (an adverse drug reaction consequent to antipsychotic exposure) could greatly improve the clinical treatment of schizophrenia but human studies are equivocal. A complementary approach is the mouse-then-human design in which a valid mouse model is used to identify susceptibility loci which are subsequently tested in human samples. We used inbred mouse strains from the Mouse Phenome Project to estimate the heritability of haloperidol-induced activity and orofacial phenotypes. 159 mice from 27 inbred strains were chronically treated with haloperidol (3 mg/kg/day via subdermal slow-release pellets) and monitored for the development of vacuous chewing movements (VCMs, the mouse analog of TD) and other movement phenotypes derived from open field activity and the inclined screen test. The test battery was assessed at 0, 30, 60, 90, and 120 days in relation to haloperidol exposure. As expected, haloperidol caused marked changes in VCMs, activity in the open field, and EPS. Unexpectedly, factor analysis demonstrated that these measures were imprecise assessments of a latent construct rather than discrete constructs. The heritability of a composite phenotype was ~0.9 after incorporation of the longitudinal nature of the design. Murine VCMs are a face valid animal model of antipsychotic-induced TD and heritability estimates from this study support the feasibility of mapping of susceptibility loci for VCMs.

Plasma fibronectin concentration in inbred mouse strains

Plasma fibronectin concentration in inbred mouse strains

Mouse segmental duplication and copy number variation.

Detailed analyses of the clone-based genome assembly reveal that the recent duplication content of mouse (4.94%) is now comparable to that of human (5.5%), in contrast to previous estimates from the whole-genome shotgun sequence assembly. However, the architecture of mouse and human genomes differs markedly: most mouse duplications are organized into discrete clusters of tandem duplications that show depletion of genes and transcripts and enrichment of long interspersed nuclear element (LINE) and long terminal repeat (LTR) retroposons. We assessed copy number variation of the C57BL/6J duplicated regions within 15 mouse strains previously used for genetic association studies, sequencing and the Mouse Phenome Project. We determined that over 60% of these base pairs are polymorphic among the strains (on average, there was 20 Mb of copy-number-variable DNA between different mouse strains). Our data suggest that different mouse strains show comparable, if not greater, copy number polymorphism when compared to human; however, such variation is more locally restricted. We show large and complex patterns of interstrain copy number variation restricted to large gene families associated with spermatogenesis, pregnancy, viviparity, pheromone signaling and immune response.

Mouse strain‐specific coding polymorphism in the Slc4a2/Ae2 gene encodes Ae2c2 variants differing in isoform‐specific dominant negative activity

The Slc4a2/Ae2 gene encodes multiple polypeptides arising from alternate promoter usage. The Ae2c promoter gives rise to only one Ae2c transcript from the human Ae2 gene, but to two, alternatively spliced, Ae2c1 and Ae2c2 transcripts from the mouse and rat genes. Unlike in the rat, the mouse Ae2c2 transcript encodes a novel Ae2c2 polypeptide. Here we report that the Ae2c2 residue 9 can be either proline or serine in a mouse strain-specific manner. Both Ae2c2 polypeptides express low function in Xenopus oocytes secondary to reduced or absent surface expression. Ae2c2S, but not Ae2c2P, exerts a dominant negative effect when coexpressed with Ae2a polypeptide, has a less prominent effect when coexpressed with Ae2b1 or Ae2c1 polypeptides, but has no effect on the function of coexpressed Ae2b2 polypeptide. Coexpression of Ae2c2P does not reduce activity of any Ae2 polypeptide variant. Ae2c2S and Ae2c2P also express low functional activity in HEK-293 cells. Knowledge of strain-specific coding polymorphisms with potential functional consequences such as that of Ae2c2 should aid in interpretation of strain-specific phenotypes investigated in the mouse phenome project.

Single nucleotide polymorphisms in mannan‐binding lectins and ficolins in various strains of mice

Mannan-binding lectin (MBL) and ficolin are collagenous lectins produced primarily by the liver and are involved in innate resistance to microbial pathogens. Mice have two MBL genes (Mbl1 and Mbl2) that encode MBL-A and MBL-C, respectively. Similarly, the murine Fcna and Fcnb genes encode ficolin-A and ficolin-B. Several single nucleotide polymorphisms (SNP) in the human MBL2 gene are responsible for various innate immune dysfunctions due to abnormal structure or expression of human MBL-C. In these studies, we identified SNPs in the expressed collagenous lectin genes Mbl1, Mbl2, Fcna, and Fcnb in 10 strains of mice designated high priority Group A strains by the Mouse Phenome Project (129S1/SvImJ, A/J, BALB/cByJ, C3H/HeJ, C57BL/6 J, DBA/2 J, FVB/NJ, SJL/J, CAST/EiJ and SPRET/EiJ) by sequencing gene exons by reverse transcription-polymerase chain reaction (RT-PCR). Sequence comparisons identified a total of 15 structural SNPs in Mbl1 in two strains, 27 SNPs in Mbl2 in five strains, and 19 and 15 SNPs in Fcna and Fcnb, respectively, in two strains. Two non-synonymous SNPs were identified in the collagen-like domain of mouse Fcnb that are similar to the coding polymorphisms in the collagen-like domain of human MBL2. Most of the non-synonymous SNPs identified in Mbl1 and Mbl2 occurred in the carbohydrate-recognition domains (CRDs), and some resulted in altered residues close to known ligand binding sites. Similarly, most non-synonymous SNPs of Fcna and Fcnb were identified in the fibrinogen-like CRD. The miscoding SNPs found in the CRD regions of mouse Mbl1, Mbl2, Fcna and Fcnb may be associated with strain differences in glycan binding avidity and disposition of microbial or host ligands. Furthermore, the non-synonymous mutations in the collagen-like domain of Fcnb may alter the structure of the mature ficolin-B protein leading to functional deficiencies. These differences may be important in the pathogenesis of susceptibility differences between inbred strains to various infectious microorganisms.

MtDNA phylogeny and evolution of laboratory mouse strains

Inbred mouse strains have been maintained for more than 100 years, and they are thought to be a mixture of four different mouse subspecies. Although genealogies have been established, female inbred mouse phylogenies remain unexplored. By a phylogenetic analysis of newly generated complete mitochondrial DNA sequence data in 16 strains, we show here that all common inbred strains descend from the same Mus musculus domesticus female wild ancestor, and suggest that they present a different mitochondrial evolutionary process than their wild relatives with a faster accumulation of replacement substitutions. Our data complement forthcoming results on resequencing of a group of priority strains, and they follow recent efforts of the Mouse Phenome Project to collect and make publicly available information on various strains.

Combined Application of Behavior Genetics and Microarray Analysis to Identify Regional Expression Themes and Gene–Behavior Associations

In this report we link candidate genes to complex behavioral phenotypes by using a behavior genetics approach. Gene expression signatures were generated for the prefrontal cortex, ventral striatum, temporal lobe, periaqueductal gray, and cerebellum in eight inbred strains from priority group A of the Mouse Phenome Project. Bioinformatic analysis of regionally enriched genes that were conserved across all strains revealed both functional and structural specialization of particular brain regions. For example, genes encoding proteins with demonstrated anti-apoptotic function were over-represented in the cerebellum, whereas genes coding for proteins associated with learning and memory were enriched in the ventral striatum, as defined by the Expression Analysis Systematic Explorer (EASE) application. Association of regional gene expression with behavioral phenotypes was exploited to identify candidate behavioral genes. Phenotypes that were investigated included anxiety, drug-naive and ethanol-induced distance traveled across a grid floor, and seizure susceptibility. Several genes within the glutamatergic signaling pathway (i.e., NMDA/glutamate receptor subunit 2C, calmodulin, solute carrier family 1 member 2, and glutamine synthetase) were identified in a phenotype-dependent and region-specific manner. In addition to supporting evidence in the literature, many of the genes that were identified could be mapped in silico to surrogate behavior-related quantitative trait loci. The approaches and data set described herein serve as a valuable resource to investigate the genetic underpinning of complex behaviors.

Mouse inbred strain differences in ethanol drinking to intoxication

Recently, we described a simple procedure, Drinking in the Dark (DID), in which C57BL/6J mice self-administer ethanol to a blood ethanol concentration (BEC) above 1 mg/ml. The test consists of replacing the water with 20% ethanol in the home cage for 4 h early during the dark phase of the light/dark cycle. Three experiments were conducted to explore this high ethanol drinking model further. In experiment 1, a microanalysis of C57BL/6J behavior showed that the pattern of ethanol drinking was different from routine water intake. In experiment 2, drinking impaired performance of C57BL/6J on the accelerating rotarod and balance beam. In experiment 3, 12 inbred strains were screened to estimate genetic influences on DID and correlations with other traits. Large, reliable differences in intake and BEC were detected among the strains, with C57BL/6J showing the highest values. Strain means were positively correlated with intake and BEC in the standard (24 h) and a limited (4 h) two-bottle ethanol vs. water test, but BECs reached higher levels for DID. Strain mean correlations with other traits in the Mouse Phenome Project database supported previously reported genetic relationships of high ethanol drinking with low chronic ethanol withdrawal severity and low ethanol-conditioned taste aversion. We extend these findings by showing that the correlation estimates remain relatively unchanged even after correcting for phylogenetic relatedness among the strains, thus relaxing the assumption that the strain means are statistically independent. We discuss applications of the model for finding genes that predispose pharmacologically significant drinking in mice.

Susceptibility to Alkylator-Induced Cancer Is a Heritable Trait.

Secondary malignancies are a serious adverse consequence of alkylator chemotherapy treatment. The incidence of secondary hematopoietic malignancies (t-MDS/AML) exceeds 11% in several recent reports. Secondary solid tumors (lung, breast) also occur after alkylator exposure. While patient age, primary diagnosis, and chemotherapy dose are established risk factors, there is increasing evidence that germline genetic factors are also important in determining risk. The genetic basis of susceptibility to alkylator-induced secondary neoplasms is poorly understood. Most studies have focused on genes in drug metabolic pathways. A limited number of genetic polymorphisms with modest impact on risk for alkylator-induced cancer have been reported. We hypothesize that other genetic contributors to alkylator-induced cancer susceptibility have not yet been identified. To screen genome-wide for novel heritable susceptibility factors, we established a mouse model of alkylator-induced malignancy. In collaboration with the Mouse Phenome Project, we exposed mice from 20 inbred strains to the prototypical alkylating agent, N-nitroso-N-ethylurea (ENU). Mice (n=12 per strain) received two doses of ENU (100mg/kg, IP) or no treatment (n=12 per strain) at 9 and 10 weeks of age. ENU was a potent carcinogen in many of the strains tested, inducing 140 tumors in 240 ENU-treated mice (66% incidence of at least one tumor in evaluable mice), compared to a background incidence of 8% spontaneous tumors in 240 strain, age, and sex-matched control mice (relative risk = 8.4, p< 0.0001). A wide variety of tumor histologies were noted, including epithelial carcinomas, soft tissue sarcomas, and hematopoietic tumors. Cancer susceptibility was a heritable trait for the most common tumor types, lung adenocarcinoma (H2 = 0.25, implying that 25% of cancer risk is heritable), T-cell lymphoma (H2 = 0.19), and myeloid malignancies (H2 = 0.10). The development of hematopoietic tumors (either lymphoid or myeloid) was negatively associated with the likelihood of developing gastrointestinal or lung adenocarcinoma. In silico quantitative trait locus (QTL) mapping was used to identify genomic intervals associated with cancer susceptibility. Strains were segregated by haplotype using a panel of 10,900 single nucleotide polymorphisms (SNPs). Differences in cancer susceptibility between haplotypes were assessed by ANOVA. After correcting for false detection, genomic intervals significantly associated with specific cancer phenotypes were identified. Preliminary analysis linked lung cancer susceptibility to four intervals on chromosomes 1,2,6, and 12. The latter interval contains Ahr, a highly polymorphic gene encoding the aryl hydrocarbon receptor previously implicated in carcinogenicity of environmental toxins. Lymphoma susceptibility was linked to two regions on chromosomes 3 and 18. The latter contains a member of the frizzled family of Wnt receptors. This novel mouse model recapitulates many features of human alkylator-associated cancer and supports the hypothesis that susceptibility to this syndrome is influenced by inherited polymorphisms that could be used to inform clinical treatment decisions.

The Mouse Phenome Project

The laboratory mouse is the organism of choice for many studies in biology and medicine. Reliable phenotypic data are essential for the full utility of genotypic information emerging from efforts to sequence human and mouse genomes. The Mouse Phenome Project has been organized to help accomplish this task by establishing a collection of baseline phenotypic data on commonly used and genetically diverse inbred mouse strains and making this information publicly available through a web-accessible database. The Mouse Phenome Database (MPD) is being developed to manage these data and to provide researchers with tools for exploring both raw phenotypic data and comparative summary analyses. The MPD serves as a repository for detailed protocols and raw data. This resource enables investigators to identify appropriate strains for (1) physiological testing, (2) drug discovery, (3) toxicology studies, (4) mutagenesis, (5) modeling human diseases, (6) QTL analyses and identification of new genes and (7) unraveling the influence of environment on genotype.

A rating scale for wildness and ease of handling laboratory mice: results for 21 inbred strains tested in two laboratories.

Rating scales for difficulty in capturing and holding mice were devised that proved to be easy to use and highly sensitive to differences among mouse strains on the A and B priority lists of the Mouse Phenome Project. The simplicity of the scales makes it feasible to rate wildness during behavioral test sessions without adding much to testing time or distracting the technician from the principal task at hand. Overall wildness and placidity ratings obtained by combining capture and hold ratings provide a good impression of the difficulties encountered while working with lab mice in the course of complex experiments. Ratings of 21 inbred strains during the course of 15 behavioral tests in two laboratories demonstrated that the SPRET/Ei, PERA/Ei, CAST/Ei and SWR/J strains were particularly difficult to handle. The NOD/LtJ strain posed no special challenge in the Edmonton laboratory but was very difficult to handle in the Portland lab. The rating scales should be useful for judging the difficulties in working with novel targeted or induced mutations in mice as well as effects of a variety of environmental treatments or drugs.

Survey of 21 inbred mouse strains in two laboratories reveals that BTBR T/+ tf/tf has severely reduced hippocampal commissure and absent corpus callosum

A morphometric survey of brain size and forebrain commissures of 21 inbred mouse strains from the Jackson Laboratory was done with animals tested in two laboratories as part of the Mouse Phenome Project. Strain BTBR T/+ tf/tf was found to have 100% total absence of the corpus callosum as well as severe reduction of the hippocampal commissure in almost every animal, the most severe commissure defect observed to date in any commercially available mouse strain. The strain 129S1/SvImJ had a milder defect with incomplete penetrance. Crosses of BTBR mice with inbred strains BALB/cWah1, 129P1/ReJ, and the recombinant strain 9XCA/Wah having a severe commissure defect supported a two-locus model of the genetic defect in these strains. Brain size varied greatly among strains but for any one strain was almost identical in mice housed for 5 weeks in the two laboratories. Sex differences in brain weight and forebrain commissure sizes were not statistically significant.

Large-scale, high-throughput screening for coagulation and hematologic phenotypes in mice.

The Mouse Phenome Project is an international effort to systematically gather phenotypic data for a defined set of inbred mouse strains. For such large-scale projects the development of high-throughput screening protocols that allow multiple tests to be performed on a single mouse is essential. Here we report hematologic and coagulation data for more than 30 inbred strains. Complete blood counts were performed using an Advia 120 analyzer. For coagulation testing, we successfully adapted the Dade Behring BCS automated coagulation analyzer for use in mice by lowering sample and reagent volume requirements. Seven automated assay procedures were developed. Small sample volume requirements make it possible to perform multiple tests on a single animal without euthanasia, while reductions in reagent volume requirements reduce costs. The data show that considerable variation in many basic hematological and coagulation parameters exists among the inbred strains. These data, freely available on the World Wide Web, allow investigators to knowledgeably select the most appropriate strain(s) to meet their individual study designs and goals.

Inbred strain variation in lung function.

The purpose of the present study was to determine the strain-specific phenotype variance of lung function parameters among common inbred laboratory mouse strains. In accordance with the "Mouse Phenome Project" run by The Jackson Laboratory (http://www.jax.org/phenome), lung volumes, lung mechanics, and diffusing capacity of 16 males and 16 females of the strains C3H/HeJ, BALB/cByJ, C57B1/6J, A/J, FVB/J, 129SV/ImJ, and SWR/J were determined in a standardized manner. The defined respiratory maneuvers for lung function testing were performed with a custom-made, computer-controlled servo-ventilator in anesthetized animals. Sex differences within the strains were found in most (83%) of the absolute lung function parameters. Usually, normalization to body or lung size completely compensates for the observed gender differences. There was great diversity between strains for all of the lung function parameters studied; for example, the total lung capacity as well as the pulmonary diffusing capacity for carbon monoxide varied by 50% and the static lung compliance by a factor of almost two among the strains. Little, but statistically significant variability was detectable for the dead space volume and the respiratory system resistance. There was no clear-cut evidence for any strain exhibiting either the smallest or the largest values for all parameters studied, suggesting that there were no simple allometric relationships of lung size between the strains. Well-established genealogical relationships among strains were not constantly reflected in phenotype similarities of pulmonary function. Therefore, these data strongly support heritable genetic traits for pulmonary function. Moreover, it constitutes a basis for further genetic lung function-related studies.

A mouse phenome project.

A community-wide effort to establish baseline phenotypic data on commonly used and genetically diverse inbred mouse strains and to provide the information through a publicly accessible database.