Systems genetics uncover new loci containing functional gene candidates in Mycobacterium tuberculosis-infected Diversity Outbred mice.

Mycobacterium tuberculosis, the bacillus that causes tuberculosis (TB), infects 2 billion people across the globe, and results in 8-9 million new TB cases and 1-1.5 million deaths each year. Most patients have no known genetic basis that predisposes them to disease. We investigated the complex genetic basis of pulmonary TB by modelling human genetic diversity with the Diversity Outbred mouse population. When infected with M. tuberculosis, one-third develop early onset, rapidly progressive, necrotizing granulomas and succumb within 60 days. The remaining develop non-necrotizing granulomas and survive longer than 60 days. Genetic mapping using clinical indicators of disease, granuloma histopathological features, and immune response traits identified five new loci on mouse chromosomes 1, 2, 4, 16 and three previously identified loci on chromosomes 3 and 17. Quantitative trait loci (QTLs) on chromosomes 1, 16, and 17, associated with multiple correlated traits and had similar patterns of allele effects, suggesting these QTLs contain important genetic regulators of responses to M. tuberculosis. To narrow the list of candidate genes in QTLs, we used a machine learning strategy that integrated gene expression signatures from lungs of M. tuberculosis-infected Diversity Outbred mice with gene interaction networks, generating functional scores. The scores were then used to rank candidates for each mapped trait in each locus, resulting in 11 candidates: Ncf2, Fam20b, S100a8, S100a9, Itgb5, Fstl1, Zbtb20, Ddr1, Ier3, Vegfa, and Zfp318. Importantly, all 11 candidates have roles in infection, inflammation, cell migration, extracellular matrix remodeling, or intracellular signaling. Further, all candidates contain single nucleotide polymorphisms (SNPs), and some but not all SNPs were predicted to have deleterious consequences on protein functions. Multiple methods were used for validation including (i) a statistical method that showed Diversity Outbred mice carrying PWH/PhJ alleles on chromosome 17 QTL have shorter survival; (ii) quantification of S100A8 protein levels, confirming predicted allele effects; and (iii) infection of C57BL/6 mice deficient for the S100a8 gene. Overall, this work demonstrates that systems genetics using Diversity Outbred mice can identify new (and known) QTLs and new functionally relevant gene candidates that may be major regulators of granuloma necrosis and acute inflammation in pulmonary TB.

Genetic background influences susceptibility to chemotherapy-induced hematotoxicity.

Hematotoxicity is a life-threatening side effect of many chemotherapy regimens. Although clinical factors influence patient responses, genetic factors may also play an important role. We sought to identify genomic loci that influence chemotherapy-induced hematotoxicity by dosing Diversity Outbred mice with one of three chemotherapy drugs; doxorubicin, cyclophosphamide or docetaxel. We observed that each drug had a distinct effect on both the changes in blood cell subpopulations and the underlying genetic architecture of hematotoxicity. For doxorubicin, we mapped the change in cell counts before and after dosing and found that alleles of ATP-binding cassette B1B (Abcb1b) on chromosome 5 influence all cell populations. For cyclophosphamide and docetaxel, we found that each cell population was influenced by distinct loci, none of which overlapped between drugs. These results suggest that susceptibility to chemotherapy-induced hematotoxicity is influenced by different genes for different chemotherapy drugs.

High-precision genetic mapping of behavioral traits in the diversity outbred mouse population.

Historically our ability to identify genetic variants underlying complex behavioral traits in mice has been limited by low mapping resolution of conventional mouse crosses. The newly developed Diversity Outbred (DO) population promises to deliver improved resolution that will circumvent costly fine-mapping studies. The DO is derived from the same founder strains as the Collaborative Cross (CC), including three wild-derived strains. Thus the DO provides more allelic diversity and greater potential for discovery compared to crosses involving standard mouse strains. We have characterized 283 male and female DO mice using open-field, light-dark box, tail-suspension and visual-cliff avoidance tests to generate 38 behavioral measures. We identified several quantitative trait loci (QTL) for these traits with support intervals ranging from 1 to 3 Mb in size. These intervals contain relatively few genes (ranging from 5 to 96). For a majority of QTL, using the founder allelic effects together with whole genome sequence data, we could further narrow the positional candidates. Several QTL replicate previously published loci. Novel loci were also identified for anxiety- and activity-related traits. Half of the QTLs are associated with wild-derived alleles, confirming the value to behavioral genetics of added genetic diversity in the DO. In the presence of wild-alleles we sometimes observe behaviors that are qualitatively different from the expected response. Our results demonstrate that high-precision mapping of behavioral traits can be achieved with moderate numbers of DO animals, representing a significant advance in our ability to leverage the mouse as a tool for behavioral genetics.

Discovery of blood transcriptomic markers for depression in animal models and pilot validation in subjects with early-onset major depression.

Early-onset major depressive disorder (MDD) is a serious and prevalent psychiatric illness in adolescents and young adults. Current treatments are not optimally effective. Biological markers of early-onset MDD could increase diagnostic specificity, but no such biomarker exists. Our innovative approach to biomarker discovery for early-onset MDD combined results from genome-wide transcriptomic profiles in the blood of two animal models of depression, representing the genetic and the environmental, stress-related, etiology of MDD. We carried out unbiased analyses of this combined set of 26 candidate blood transcriptomic markers in a sample of 15-19-year-old subjects with MDD (N=14) and subjects with no disorder (ND, N=14). A panel of 11 blood markers differentiated participants with early-onset MDD from the ND group. Additionally, a separate but partially overlapping panel of 18 transcripts distinguished subjects with MDD with or without comorbid anxiety. Four transcripts, discovered from the chronic stress animal model, correlated with maltreatment scores in youths. These pilot data suggest that our approach can lead to clinically valid diagnostic panels of blood transcripts for early-onset MDD, which could reduce diagnostic heterogeneity in this population and has the potential to advance individualized treatment strategies.

Gene expression patterns in the hippocampus and amygdala of endogenous depression and chronic stress models.

The etiology of depression is still poorly understood, but two major causative hypotheses have been put forth: the monoamine deficiency and the stress hypotheses of depression. We evaluate these hypotheses using animal models of endogenous depression and chronic stress. The endogenously depressed rat and its control strain were developed by bidirectional selective breeding from the Wistar-Kyoto (WKY) rat, an accepted model of major depressive disorder (MDD). The WKY More Immobile (WMI) substrain shows high immobility/despair-like behavior in the forced swim test (FST), while the control substrain, WKY Less Immobile (WLI), shows no depressive behavior in the FST. Chronic stress responses were investigated by using Brown Norway, Fischer 344, Lewis and WKY, genetically and behaviorally distinct strains of rats. Animals were either not stressed (NS) or exposed to chronic restraint stress (CRS). Genome-wide microarray analyses identified differentially expressed genes in hippocampi and amygdalae of the endogenous depression and the chronic stress models. No significant difference was observed in the expression of monoaminergic transmission-related genes in either model. Furthermore, very few genes showed overlapping changes in the WMI vs WLI and CRS vs NS comparisons, strongly suggesting divergence between endogenous depressive behavior- and chronic stress-related molecular mechanisms. Taken together, these results posit that although chronic stress may induce depressive behavior, its molecular underpinnings differ from those of endogenous depression in animals and possibly in humans, suggesting the need for different treatments. The identification of novel endogenous depression-related and chronic stress response genes suggests that unexplored molecular mechanisms could be targeted for the development of novel therapeutic agents.

Naive application of permutation testing leads to inflated type I error rates.

Failure to account for family structure within populations or in complex mating designs via uninformed applications of permutation testing will lead to inflated type I error rates. Careful consideration of the design factors is essential since some situations allow several valid permutation strategies, and the choice that maximizes statistical power will not always be intuitive.

Identification of quantitative trait loci for locomotor activation and anxiety using closely related inbred strains.

We carried out a quantitative trait loci (QTL) mapping experiment in two phenotypically similar inbred mouse strains, C57BL/6J and C58/J, using the open-field assay, a well-established model of anxiety-related behavior in rodents. This intercross was initially carried out as a control cross for an ethylnitrosurea mutagenesis mapping study. Surprisingly, although open-field behavior is similar in the two strains, we identified significant QTL in their F2 progeny. Marker regression identified a locus on Chr 8 having associations with multiple open-field measures and a significant interaction between loci on Chr 13 and 17. Together, the Chr 8 locus and the interaction effect form the core set of QTL controlling these behaviors with additional loci on Chr 1 and 6 present in a subset of the behaviors.

Overexpression of innate immune response genes in a model of recessive polycystic kidney disease.

Defects in the primary cilium/basal body complex of renal tubular cells cause polycystic kidney disease (PKD). To uncover pathways associated with disease progression, we determined the kidney transcriptome of 10-day-old severely and mildly affected cpk mice, a model of recessive PKD. In the severe phenotype, the most highly expressed genes were those associated with the innate immune response including many macrophage markers, particularly those associated with a profibrotic alternative activation pathway. Additionally, gene expression of macrophage activators was dominated by the complement system factors including the central complement component 3. Additional studies confirmed increased complement component 3 protein levels in both cystic and non-cystic epithelia in the kidneys of cpk compared to wild-type mice. We also found elevated complement component 3 activation in two other mouse-recessive models and human-recessive PKD. Our results suggest that abnormal complement component 3 activation is a key element of progression in PKD.

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) by rosiglitazone suppresses components of the insulin-like growth factor regulatory system in vitro and in vivo.

Rosiglitazone (Rosi) belongs to the class of thiazolidinediones (TZDs) that are ligands for peroxisome proliferator-activated receptor gamma (PPARgamma). Stimulation of PPARgamma suppresses bone formation and enhances marrow adipogenesis. We hypothesized that activation of PPARgamma down-regulates components of the IGF regulatory system, leading to impaired osteoblast function. Rosi treatment (1 microm) of a marrow stromal cell line (UAMS-33) transfected with empty vector (U-33/c) or with PPARgamma2 (U-33/gamma2) were analyzed by microarray. Rosi reduced IGF-I, IGF-II, IGFBP-4, and the type I and II IGF receptor (IGF1R and IGF2R) expression at 72 h in U-33/gamma2 compared with U-33/c cells (P < 0.01); these findings were confirmed by RT-PCR. Rosi reduced secreted IGF-I from U-33/gamma2 cells by 75% (P < 0.05). Primary marrow stromal cells (MSCs) extracted from adult (8 months) and old (24 months) C57BL/6J (B6) mice were treated with Rosi (1 microm) for 48 h. IGF-I, IGFBP-4, and IGF1R transcripts were reduced in Rosi-treated MSCs compared with vehicle (P < 0.01) and secreted IGF-I was also suppressed (P < 0.05). B6 mice treated with Rosi (20 mg/kg.d) for short duration (i.e. 4 d), and long term (i.e. 7 wk) had reduced serum IGF-I; this was accompanied by markedly suppressed IGF-I transcripts in the liver and peripheral fat of treated animals. To determine whether Rosi affected circulating IGF-I in humans, we measured serum IGF-I, IGFBP-2, and IGFBP-3 at four time points in 50 postmenopausal women randomized to either Rosi (8 mg/d) or placebo. Rosi-treated subjects had significantly lower IGF-I at 8 wk than baseline (-25%, P < 0.05), and at 16 wk their levels were reduced 14% vs. placebo (P = 0.15). We conclude that Rosi suppresses IGF-I expression in bone and liver; these changes could affect skeletal acquisition through endocrine and paracrine pathways.

PPARgamma2 Regulates a Molecular Signature of Marrow Mesenchymal Stem Cells.

Bone formation and hematopoiesis are anatomically juxtaposed and share common regulatory mechanisms. Bone marrow mesenchymal stromal/stem cells (MSC) contain a compartment that provides progeny with bone forming osteoblasts and fat laden adipocytes as well as fibroblasts, chondrocytes, and muscle cells. In addition, marrow MSC provide an environment for support of hematopoiesis, including the development of bone resorbing osteoclasts. The PPARgamma2 nuclear receptor is an adipocyte-specific transcription factor that controls marrow MSC lineage allocation toward adipocytes and osteoblasts. Increased expression of PPARgamma2 with aging correlates with changes in the MSC status in respect to both their intrinsic differentiation potential and production of signaling molecules that contribute to the formation of a specific marrow micro-environment. Here, we investigated the effect of PPARgamma2 on MSC molecular signature in respect to the expression of gene markers associated exclusively with stem cell phenotype, as well as genes involved in the formation of a stem cell supporting marrow environment. We found that PPARgamma2 is a powerful modulator of stem cell-related gene expression. In general, PPARgamma2 affects the expression of genes specific for the maintenance of stem cell phenotype, including LIF, LIF receptor, Kit ligand, SDF-1, Rex-1/Zfp42, and Oct-4. Moreover, the antidiabetic PPARgamma agonist TZD rosiglitazone specifically affects the expression of "stemness" genes, including ABCG2, Egfr, and CD44. Our data indicate that aging and anti-diabetic TZD therapy may affect mesenchymal stem cell phenotype through modulation of PPARgamma2 activity. These observations may have important therapeutic consequences and indicate a need for more detailed studies of PPARgamma2 role in stem cell biology.

A major quantitative trait locus on chromosome 3 controls colitis severity in IL-10-deficient mice.

Colitic lesions are much more severe in C3H/HeJBir (C3H) than C57BL/6J (B6) mice after 10 backcrosses of a disrupted interleukin-10 (Il10) gene. This study identified cytokine deficiency-induced colitis susceptibility (Cdcs) modifiers by using quantitative trait locus (QTL) analysis. A segregating F(2) population (n = 408) of IL-10-deficient mice was genotyped and necropsied at 6 weeks of age. A major C3H-derived colitogenic QTL (Cdcs1) on chromosome (Chr.) 3 contributed to lesions in both cecum [logarithm of odds ratio (LOD) = 14.6)] and colon (LOD = 26.5) as well as colitis-related phenotypes such as spleen/body weight ratio, mesenteric lymph node/body weight ratio, and secretory IgA levels. Evidence for other C3H QTL on Chr. 1 (Cdcs2) and Chr. 2 (Cdcs3) was obtained. Cdcs1 interacted epistatically or contributed additively with loci on other chromosomes. The resistant B6 background also contributed colitogenic QTL: Cdcs4 (Chr. 8), Cdcs5 (Chr. 17, MHC), and Cdcs6 (Chr. 18). Epistatic interactions between B6 QTL on Chr. 8 and 18 contributing to cecum hyperplasia were particularly striking. In conclusion, a colitogenic susceptibility QTL on Chr. 3 has been shown to exacerbate colitis in combination with modifiers contributed from both parental genomes. The complex nature of interactions among loci in this mouse model system, coupled with separate deleterious contributions from both parental strains, illustrates why detection of human inflammatory bowel disease linkages has proven to be so difficult. A human ortholog of the Chr. 3 QTL, if one exists, would map to Chr. 4q or 1p.

A statistical framework for quantitative trait mapping.

We describe a general statistical framework for the genetic analysis of quantitative trait data in inbred line crosses. Our main result is based on the observation that, by conditioning on the unobserved QTL genotypes, the problem can be split into two statistically independent and manageable parts. The first part involves only the relationship between the QTL and the phenotype. The second part involves only the location of the QTL in the genome. We developed a simple Monte Carlo algorithm to implement Bayesian QTL analysis. This algorithm simulates multiple versions of complete genotype information on a genomewide grid of locations using information in the marker genotype data. Weights are assigned to the simulated genotypes to capture information in the phenotype data. The weighted complete genotypes are used to approximate quantities needed for statistical inference of QTL locations and effect sizes. One advantage of this approach is that only the weights are recomputed as the analyst considers different candidate models. This device allows the analyst to focus on modeling and model comparisons. The proposed framework can accommodate multiple interacting QTL, nonnormal and multivariate phenotypes, covariates, missing genotype data, and genotyping errors in any type of inbred line cross. A software tool implementing this procedure is available. We demonstrate our approach to QTL analysis using data from a mouse backcross population that is segregating multiple interacting QTL associated with salt-induced hypertension.

Quantitative trait loci for femoral and lumbar vertebral bone mineral density in C57BL/6J and C3H/HeJ inbred strains of mice.

Significant differences in vertebral (9%) and femoral (50%) adult bone mineral density (BMD) between the C57BL/6J (B6) and C3H/HeJ (C3H) inbred strains of mice have been subjected to genetic analyses for quantitative trait loci (QTL). Nine hundred eighty-six B6C3F2 females were analyzed to gain insight into the number of genes that regulate peak BMD and their locations. Femurs and lumbar vertebrae were isolated from 4-month-old B6C3F2 females at skeletal maturity and then BMD was determined by peripheral quantitative computed tomography (pQCT). Estimates of BMD heritability were 83% for femurs and 72% for vertebrae. Genomic DNA from F2 progeny was screened for 107 polymerase chain reaction (PCR)-based markers discriminating B6 and C3H alleles on all 19 autosomes. The regression analyses of markers on BMD revealed ten chromosomes (1, 2, 4, 6, 11, 12, 13, 14, 16, and 18) carrying QTLs for femurs and seven chromosomes (1, 4, 7, 9, 11, 14, and 18) carrying QTLs for vertebrae, each with log10 of the odds ratio (LOD) scores of 2.8 or better. The QTLs on chromosomes (Chrs) 2, 6, 12, 13, and 16 were unique to femurs, whereas the QTLs on Chrs 7 and 9 were unique to vertebrae. When the two bone sites had a QTL on the same chromosome, the same marker had the highest, although different, LOD score. A pairwise comparison by analysis of variance (ANOVA) did not reveal significant gene x gene interactions between QTLs for either bone site. BMD variance accounted for by individual QTLs ranged from 1% to 10%. Collectively, the BMD QTLs for femurs accounted for 35.1% and for vertebrae accounted for 23.7 % of the F2 population variances in these bones. When mice were homozygous c3/c3 in the QTL region, 8 of the 10 QTLs increased, while the remaining two QTLs on Chrs 6 and 12 decreased, femoral BMD. Similarly, when mice were homozygous c3/c3 in the QTL region for the vertebrae, five of the seven QTLs increased, while two QTLs on Chrs 7 and 9 decreased, BMD. These findings show the genetic complexity of BMD with multiple genes participating in its regulation. Although 5 of the 12 QTLs are considered to be skeleton-wide loci and commonly affect both femurs and vertebrae, each of the bone sites also exhibited unique QTLs. Thus, the BMD phenotype can be partitioned into its genetic components and the effects of these loci on normal bone biology can be determined. Importantly, the BMD QTLs that we have identified are in regions of the mouse genome that have known human homology, and the QTLs will become useful experimental tools for mechanistic and therapeutic analyses of bone regulatory genes.

Bootstrapping cluster analysis: assessing the reliability of conclusions from microarray experiments.

We introduce a general technique for making statistical inference from clustering tools applied to gene expression microarray data. The approach utilizes an analysis of variance model to achieve normalization and estimate differential expression of genes across multiple conditions. Statistical inference is based on the application of a randomization technique, bootstrapping. Bootstrapping has previously been used to obtain confidence intervals for estimates of differential expression for individual genes. Here we apply bootstrapping to assess the stability of results from a cluster analysis. We illustrate the technique with a publicly available data set and draw conclusions about the reliability of clustering results in light of variation in the data. The bootstrapping procedure relies on experimental replication. We discuss the implications of replication and good design in microarray experiments.

Genetic analysis of a new mouse model for non-insulin-dependent diabetes.

The TallyHo (TH) mouse strain is a newly established model for non-insulin-dependent diabetes mellitus (NIDDM). TH mice show obesity, hyperinsulinemia, hyperlipidemia, and male-limited hyperglycemia. A genetic dissection of the diabetes syndrome has been carried out using male backcross 1 progeny obtained from crosses between (C57BL/6J x TH)F1 and TH mice or (CAST/Ei x TH)F1 and TH mice. A genome-wide scan reveals three quantitative trait loci (QTLs), Tanidd1-3 (TH-associated NIDDM) linked to hyperglycemia. The major QTL (common in both crosses), Tanidd1, maps to chromosome (Chr) 19. Additionally, gene-gene interactions contributing to hyperglycemia have been observed between Tanidd1 and a locus on Chr 18 as well as between Tanidd2 and a locus on Chr 16. The overt hyperglycemia in TH mice is, therefore, likely due to a mutation in a major diabetes susceptibility locus on Chr 19, which interacts with additional genes to lead to an observable phenotype.

Statistical design and the analysis of gene expression microarray data.

Gene expression microarrays are an innovative technology with enormous promise to help geneticists explore and understand the genome. Although the potential of this technology has been clearly demonstrated, many important and interesting statistical questions persist. We relate certain features of microarrays to other kinds of experimental data and argue that classical statistical techniques are appropriate and useful. We advocate greater attention to experimental design issues and a more prominent role for the ideas of statistical inference in microarray studies.

Genome-wide epistatic interaction analysis reveals complex genetic determinants of circadian behavior in mice.

Genetic heterogeneity underlies many phenotypic variations observed in circadian rhythmicity. Continuous distributions in measures of circadian behavior observed among multiple inbred strains of mice suggest that the inherent contributions to variability are polygenic in nature. To identify genetic loci that underlie this complex behavior, we have carried out a genome-wide complex trait analysis in 196 (C57BL/6J X BALB/cJ)F(2) hybrid mice. We have characterized variation in this panel of F(2) mice among five circadian phenotypes: free-running circadian period, phase angle of entrainment, amplitude of the circadian rhythm, circadian activity level, and dissociation of rhythmicity. Our genetic analyses of these phenotypes have led to the identification of 14 loci having significant effects on this behavior, including significant main effect loci that contribute to three of these phenotypic measures: period, phase, and amplitude. We describe an additional locus detection method, genome-wide genetic interaction analysis, developed to identify locus pairs that may interact epistatically to significantly affect phenotype. Using this analysis, we identified two additional pairs of loci that have significant effects on dissociation and activity level; we also detected interaction effects in loci contributing to differences of period, phase, and amplitude. Although single gene mutations can affect circadian rhythms, the analysis of interstrain variants demonstrates that significant genetic complexity underlies this behavior. Importantly, most of the loci that we have detected by these methods map to locations that differ from the nine known clock genes, indicating the presence of additional clock-relevant genes in the mammalian circadian system. These data demonstrate the analytical value of both genome-wide complex trait and epistatic interaction analyses in further understanding complex phenotypes, and point to promising approaches for genetic analysis of such phenotypes in other mammals, including humans.

The impact of reduced frequency of cage changes on the health of mice housed in ventilated cages.

Our purpose in this investigation was to determine if we could reduce cage changing frequency without adversely affecting the health of mice. We housed mice at three different cage changing frequencies: 7, 14, and 21 days, each at three different cage ventilation rates: 30, 60 and 100 air changes per hour (ACH), for a total of nine experimental conditions. For each condition, we evaluated the health of 12 breeding pairs and 12 breeding trios of C57BL/6J mice for 7 months. Health was assessed by breeding performance, weanling weight and growth, plasma corticosterone levels, immune function, and histological examination of selected organs. Over a period of 4 months, we monitored the cage microenvironment for ammonia and carbon dioxide concentrations, relative humidity, and temperature one day prior to changing the cage. The relative humidity, carbon dioxide concentrations, and temperature of the cages at all conditions were within acceptable levels. Ammonia concentrations remained below 25 ppm (parts per million) in most cages, but, even at higher concentrations, did not adversely affect the health of mice. Frequency of cage changing had only one significant effect; pup mortality with pair matings was greater at the cage changing frequency of 7 days compared with 14 or 21 days. In addition, pup mortality with pair matings was higher at 30 ACH compared with other ventilation rates. In conclusion, under the conditions of this study, cage changes once every 14 days and ventilation rates of 60 ACH provide optimum conditions for animal health and practical husbandry.

Concordance of murine quantitative trait loci for salt-induced hypertension with rat and human loci.

To investigate the genetic control of salt-induced hypertension, we performed a quantitative trait locus analysis on male mice from a reciprocal backcross between the salt-sensitive C57BL/6J and the normotensive A/J inbred mouse strains after they were provided with water containing 1% salt for 2 weeks. Genome-wide scans performed on these mice and analyzed with a combination of conventional marker-based regressions and a novel simultaneous search for pairs revealed six significant quantitative trait loci associated with salt-induced blood pressure, two of which were interacting loci. These six loci, named Bpq1-6 for blood pressure quantitative trait loci, mapped to D1Mit334, D1Mit14, D4Mit164, D5Mit31, D6Mit15, and D15Mit13. Furthermore, five of these six loci were concordant with hypertension loci in rats, and four were concordant with hypertension loci in humans, suggesting that quantitative trait loci mapping in model organisms can be used to guide the search for human blood pressure genes.