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1.  Rapid genotype imputation from sequence without reference panels 
Nature genetics  2016;48(8):965-969.
Inexpensive genotyping methods are essential for genetic studies requiring large sample sizes. In human studies, array-based microarrays and high-density haplotype reference panels allow efficient genotype imputation for this purpose. However, these resources are typically unavailable in non-human settings. Here we describe a method (STITCH) for imputation based only on sequencing read data, without requiring additional reference panels or array data. We demonstrate its applicability even in settings of extremely low sequencing coverage, by accurately imputing 5.7 million SNPs at a mean r2 of 0.98 in 2,073 outbred laboratory mice (0.15X sequencing coverage). In a sample of 11,670 Han Chinese (1.7X), we achieve accuracy similar to alternative approaches that require a reference panel, demonstrating that this approach can work for genetically diverse populations. Our method enables straightforward progression from low-coverage sequence to imputed genotypes, overcoming barriers that at present restrict the application of genome-wide association study technology outside humans.
PMCID: PMC4966640  PMID: 27376236
2.  Genome-wide association of multiple complex traits in outbred mice by ultra low-coverage sequencing 
Nature genetics  2016;48(8):912-918.
Two bottlenecks impeding the genetic analysis of complex traits in rodents are access to mapping populations able to deliver gene-level mapping resolution, and the need for population specific genotyping arrays and haplotype reference panels. Here we combine low coverage sequencing (0.15X) with a novel method to impute the ancestral haplotype space in 1,887 commercially available outbred mice. We mapped 156 unique quantitative trait loci for 92 phenotypes at 5% false discovery rate. Gene-level mapping resolution was achieved at about a fifth of loci, implicating Unc13c and Pgc1-alpha at loci for the quality of sleep, Adarb2 for home cage activity, Rtkn2 for intensity of reaction to startle, Bmp2 for wound healing, Il15 and Id2 for several T-cell measures and Prkca for bone mineral content. These findings have implications for diverse areas of mammalian biology and demonstrate how GWAS can be extended via low-coverage sequencing to species with highly recombinant outbred populations.
PMCID: PMC4966644  PMID: 27376238
4.  Dissecting the Effect of Genetic Variation on the Hepatic Expression of Drug Disposition Genes across the Collaborative Cross Mouse Strains 
Frontiers in Genetics  2016;7:172.
A central challenge in pharmaceutical research is to investigate genetic variation in response to drugs. The Collaborative Cross (CC) mouse reference population is a promising model for pharmacogenomic studies because of its large amount of genetic variation, genetic reproducibility, and dense recombination sites. While the CC lines are phenotypically diverse, their genetic diversity in drug disposition processes, such as detoxification reactions, is still largely uncharacterized. Here we systematically measured RNA-sequencing expression profiles from livers of 29 CC lines under baseline conditions. We then leveraged a reference collection of metabolic biotransformation pathways to map potential relations between drugs and their underlying expression quantitative trait loci (eQTLs). By applying this approach on proximal eQTLs, including eQTLs acting on the overall expression of genes and on the expression of particular transcript isoforms, we were able to construct the organization of hepatic eQTL-drug connectivity across the CC population. The analysis revealed a substantial impact of genetic variation acting on drug biotransformation, allowed mapping of potential joint genetic effects in the context of individual drugs, and demonstrated crosstalk between drug metabolism and lipid metabolism. Our findings provide a resource for investigating drug disposition in the CC strains, and offer a new paradigm for integrating biotransformation reactions to corresponding variations in DNA sequences.
PMCID: PMC5050206  PMID: 27761138
collaborative cross mouse strains; hepatic drug disposition; eQTLs analysis; transcript isoforms; genetic variation
5.  A multiple phenotype imputation method for genetic studies 
Nature genetics  2016;48(4):466-472.
Genetic association studies have yielded a wealth of biologic discoveries. However, these have mostly analyzed one trait and one SNP at a time, thus failing to capture the underlying complexity of these datasets. Joint genotype-phenotype analyses of complex, high-dimensional datasets represent an important way to move beyond simple GWAS with great potential. The move to high-dimensional phenotypes will raise many new statistical problems. In this paper we address the central issue of missing phenotypes in studies with any level of relatedness between samples. We propose a multiple phenotype mixed model and use a computationally efficient variational Bayesian algorithm to fit the model. On a variety of simulated and real datasets from a range of organisms and trait types, we show that our method outperforms existing state-of-the-art methods from the statistics and machine learning literature and can boost signals of association.
PMCID: PMC4817234  PMID: 26901065
6.  Transcriptome Profiling in Rat Inbred Strains and Experimental Cross Reveals Discrepant Genetic Architecture of Genome-Wide Gene Expression 
G3: Genes|Genomes|Genetics  2016;6(11):3671-3683.
To test the impact of genetic heterogeneity on cis- and trans-mediated mechanisms of gene expression regulation, we profiled the transcriptome of adipose tissue in 20 inbred congenic strains derived from diabetic Goto–Kakizaki (GK) rats and Brown–Norway (BN) controls, which contain well-defined blocks (1–183 Mb) of genetic polymorphisms, and in 123 genetically heterogeneous rats of an (GK × BN)F2 offspring. Within each congenic we identified 73–1351 differentially expressed genes (DEGs), only 7.7% of which mapped within the congenic blocks, and which may be regulated in cis. The remainder localized outside the blocks, and therefore must be regulated in trans. Most trans-regulated genes exhibited approximately twofold expression changes, consistent with monoallelic expression. Altered biological pathways were replicated between congenic strains sharing blocks of genetic polymorphisms, but polymorphisms at different loci also had redundant effects on transcription of common distant genes and pathways. We mapped 2735 expression quantitative trait loci (eQTL) in the F2 cross, including 26% predominantly cis-regulated genes, which validated DEGs in congenic strains. A hotspot of >300 eQTL in a 10 cM region of chromosome 1 was enriched in DEGs in a congenic strain. However, many DEGs among GK, BN and congenic strains did not replicate as eQTL in F2 hybrids, demonstrating distinct mechanisms of gene expression when alleles segregate in an outbred population or are fixed homozygous across the entire genome or in short genomic regions. Our analysis provides conceptual advances in our understanding of the complex architecture of genome expression and pathway regulation, and suggests a prominent impact of epistasis and monoallelic expression on gene transcription.
PMCID: PMC5100866  PMID: 27646706
epistasis; Goto–Kakizaki; diabetes mellitus; quantitative trait locus; eQTL
7.  A Genome-Wide Association Study for Regulators of Micronucleus Formation in Mice 
G3: Genes|Genomes|Genetics  2016;6(8):2343-2354.
In mammals the regulation of genomic instability plays a key role in tumor suppression and also controls genome plasticity, which is important for recombination during the processes of immunity and meiosis. Most studies to identify regulators of genomic instability have been performed in cells in culture or in systems that report on gross rearrangements of the genome, yet subtle differences in the level of genomic instability can contribute to whole organism phenotypes such as tumor predisposition. Here we performed a genome-wide association study in a population of 1379 outbred Crl:CFW(SW)-US_P08 mice to dissect the genetic landscape of micronucleus formation, a biomarker of chromosomal breaks, whole chromosome loss, and extranuclear DNA. Variation in micronucleus levels is a complex trait with a genome-wide heritability of 53.1%. We identify seven loci influencing micronucleus formation (false discovery rate <5%), and define candidate genes at each locus. Intriguingly at several loci we find evidence for sexual dimorphism in micronucleus formation, with a locus on chromosome 11 being specific to males.
PMCID: PMC4978889  PMID: 27233670
Micronuclei; genomic instability; genome-wide association study (GWAS); Outbred mice; genetic mapping
8.  Multiple reference genomes and transcriptomes for Arabidopsis thaliana 
Nature  2011;477(7365):419-423.
Genetic differences between Arabidopsis thaliana accessions underlie the plant’s extensive phenotypic variation, and until now these have been interpreted largely in the context of the annotated reference accession Col-0. Here we report the sequencing, assembly and annotation of the genomes of 18 natural A. thaliana accessions, and their transcriptomes. When assessed on the basis of the reference annotation, one-third of protein-coding genes are predicted to be disrupted in at least one accession. However, re-annotation of each genome revealed that alternative gene models often restore coding potential. Gene expression in seedlings differed for nearly half of expressed genes and was frequently associated with cis variants within 5 kilobases, as were intron retention alternative splicing events. Sequence and expression variation is most pronounced in genes that respond to the biotic environment. Our data further promote evolutionary and functional studies in A. thaliana, especially the MAGIC genetic reference population descended from these accessions.
PMCID: PMC4856438  PMID: 21874022
9.  Genetic analysis of intestinal polyp development in Collaborative Cross mice carrying the ApcMin/+ mutation 
BMC Genetics  2016;17:46.
Colorectal cancer is an abnormal tissue development in the colon or rectum. Most of CRCs develop due to somatic mutations, while only a small proportion is caused by inherited mutations. Familial adenomatous polyposis is an inherited genetic disease, which is characterized by colorectal polyps. It is caused by inactivating mutations in the Adenomatous polyposis coli gene. Mice carrying and non-sense mutation in Adenomatous polyposis coli gene at site R850, which designated ApcR850X/+ (Min), develop intestinal adenomas, while the bulk of the disease is in the small intestine. A number of genetic modifier loci of Min have been mapped, but so far most of the underlying genes have not been identified. In our previous studies, we have shown that Collaborative Cross mice are a powerful tool for mapping loci responsible for phenotypic variation. As a first step towards identification of novel modifiers of Min, we assessed the phenotypic variation between 27 F1 crosses between different Collaborative cross mice and C57BL/6-Min lines.
Here, C57BL/6-Min male mice were mated with females from 27 Collaborative cross lines. F1 offspring were terminated at 23 weeks old and multiple phenotypes were collected: polyp counts, intestine length, intestine weight, packed cell volume and spleen weight. Additionally, in eight selected F1 Collaborative cross-C57BL/6-Min lines, body weight was monitored and compared to control mice carry wildtype Adenomatous polyposis coli gene. We found significant (p < 0.05) phenotypic variation between the 27 F1 Collaborative cross-C57BL/6-Min lines for all the tested phenotypes, and sex differences with traits; Colon, body weight and intestine length phenotypes, only. Heritability calculation showed that these phenotypes are mainly controlled by genetic factors.
Variation in polyp development is controlled, an appreciable extent, by genetic factors segregating in the Collaborative cross population and suggests that it is suited for identifying modifier genes associated with ApcMin/+ mutation, after assessing sufficient number of lines for quantitative trait loci analysis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12863-016-0349-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4761170  PMID: 26896154
ApcMin/+; Colorectal cancer; Collaborative cross; Familial adenomatous polyposis; Genetic modifier; Moms; Phenotyping; Recombinant Inbred lines
10.  High-fat-diet induced development of increased fasting glucose levels and impaired response to intraperitoneal glucose challenge in the collaborative cross mouse genetic reference population 
BMC Genetics  2016;17:10.
The prevalence of Type 2 Diabetes (T2D) mellitus in the past decades, has reached epidemic proportions. Several lines of evidence support the role of genetic variation in the pathogenesis of T2D and insulin resistance. Elucidating these factors could contribute to developing new medical treatments and tools to identify those most at risk. The aim of this study was to characterize the phenotypic response of the Collaborative Cross (CC) mouse genetic resource population to high-fat diet (HFD) induced T2D-like disease to evluate its suitability for this purpose.
We studied 683 mice of 21 different lines of the CC population. Of these, 265 mice (149 males and 116 females) were challenged by HFD (42 % fat); and 384 mice (239 males and145 females) of 17 of the 21 lines were reared as control group on standard Chow diet (18 % fat). Briefly, 8 week old mice were maintained on HFD until 20 weeks of age, and subsequently assessed by intraperitoneal glucose tolerance test (IPGTT). Biweekly body weight (BW), body length (BL), waist circumstance (WC), and body mass index (BMI) were measured. On statistical analysis, trait measurements taken at 20 weeks of age showed significant sex by diet interaction across the different lines and traits. Consequently, males and females were analyzed, separately. Differences among lines were analyzed by ANOVA and shown to be significant (P <0.05), for BW, WC, BMI, fasting blood glucose, and IPGTT-AUC. We use these data to infer broad sense heritability adjusted for number of mice tested in each line; coefficient of genetic variation; genetic correlations between the same trait in the two sexes, and phenotypic correlations between different traits in the same sex.
These results are consistent with the hypothesis that host susceptibility to HFD-induced T2D is a complex trait and controlled by multiple genetic factors and sex, and that the CC population can be a powerful tool for genetic dissection of this trait.
Electronic supplementary material
The online version of this article (doi:10.1186/s12863-015-0321-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4700737  PMID: 26728312
Type 2 diabetes (T2D); Metabolic syndrome; Obesity; High fat diet (HFD); Sex effects; Collaborative Cross mouse reference population; Heritability; Coefficient of genetic variation
11.  Determining Phenological Patterns Associated with the Onset of Senescence in a Wheat MAGIC Mapping Population 
The appropriate timing of developmental transitions is critical for adapting many crops to their local climatic conditions. Therefore, understanding the genetic basis of different aspects of phenology could be useful in highlighting mechanisms underpinning adaptation, with implications in breeding for climate change. For bread wheat (Triticum aestivum), the transition from vegetative to reproductive growth, the start and rate of leaf senescence and the relative timing of different stages of flowering and grain filling all contribute to plant performance. In this study we screened under Smart house conditions a large, multi-founder “NIAB elite MAGIC” wheat population, to evaluate the genetic elements that influence the timing of developmental stages in European elite varieties. This panel of recombinant inbred lines was derived from eight parents that are or recently have been grown commercially in the UK and Northern Europe. We undertook a detailed temporal phenotypic analysis under Smart house conditions of the population and its parents, to try to identify known or novel Quantitative Trait Loci associated with variation in the timing of key phenological stages in senescence. This analysis resulted in the detection of QTL interactions with novel traits such the time between “half of ear emergence above flag leaf ligule” and the onset of senescence at the flag leaf as well as traits associated with plant morphology such as stem height. In addition, strong correlations between several traits and the onset of senescence of the flag leaf were identified. This work establishes the value of systematically phenotyping genetically unstructured populations to reveal the genetic architecture underlying morphological variation in commercial wheat.
PMCID: PMC5075575  PMID: 27822218
wheat; senescence; data science; phenology; phenotyping; MAGIC
12.  Genetic Control over mtDNA and Its Relationship to Major Depressive Disorder 
Current Biology  2015;25(24):3170-3177.
Control over the number of mtDNA molecules per cell appears to be tightly regulated, but the mechanisms involved are largely unknown. Reversible alterations in the amount of mtDNA occur in response to stress suggesting that control over the amount of mtDNA is involved in stress-related diseases including major depressive disorder (MDD). Using low-coverage sequence data from 10,442 Chinese women to compute the normalized numbers of reads mapping to the mitochondrial genome as a proxy for the amount of mtDNA, we identified two loci that contribute to mtDNA levels: one within the TFAM gene on chromosome 10 (rs11006126, p value = 8.73 × 10−28, variance explained = 1.90%) and one over the CDK6 gene on chromosome 7 (rs445, p value = 6.03 × 10−16, variance explained = 0.50%). Both loci replicated in an independent cohort. CDK6 is thus a new molecule involved in the control of mtDNA. We identify increased rates of heteroplasmy in women with MDD, and show from an experimental paradigm using mice that the increase is likely due to stress. Furthermore, at least one heteroplasmic variant is significantly associated with changes in the amount of mtDNA (position 513, p value = 3.27 × 10−9, variance explained = 0.48%) suggesting site-specific heteroplasmy as a possible link between stress and increase in amount of mtDNA. These findings indicate the involvement of mitochondrial genome copy number and sequence in an organism’s response to stress.
•Loci near TFAM and CDK6 contribute to variation in the amount of mtDNA•Mutations accumulate in the mtDNA of cases of major depression•Animal experiments show that heteroplasmy can be induced by chronic stress•Amount of mtDNA is associated with site-specific heteroplasmy
Having previously shown that cases of major depressive disorder have increased mtDNA, that exposure to stress is likely responsible, and that the changes in amount of mtDNA are reversible, Cai et al. investigate in this paper the genetic control and consequences of this change and how this relates to major depressive disorder.
PMCID: PMC4691240  PMID: 26687620
13.  Collaborative cross mice in a genetic association study reveal new candidate genes for bone microarchitecture 
BMC Genomics  2015;16:1013.
The microstructure of trabecular bone is a composite trait governed by a complex interaction of multiple genetic determinants. Identifying these genetic factors should significantly improve our ability to predict of osteoporosis and its associated risks. Genetic mapping using collaborative cross mice (CC), a genetically diverse recombinant inbred mouse reference panel, offers a powerful tool to identify causal loci at a resolution under one mega base-pairs, with a relatively small cohort size.
Here, we utilized 31 CC lines (160 mice of both sexes in total) to perform genome-wide haplotype mapping across 77,808 single-nucleotide polymorphisms (SNPs). Haplotype scans were refined by imputation with the catalogue of sequence variation segregating in the CC to suggest potential candidate genes. Trabecular traits were obtained following microtomographic analysis, performed on 10-μm resolution scans of the femoral distal metaphysis. We measured the trabecular bone volume fraction (BV/TV), number (Tb.N), thickness (Tb.Th), and connectivity density (Conn.D).
Heritability of these traits ranged from 0.6 to 0.7. In addition there was a significant (P < 0.01) sex effect in all traits except Tb.Th. Our haplotype scans yielded six quantitative trait loci (QTL) at 1 % false discovery rate; BV/TV and Tb.Th produced two proximal loci each, on chromosome 2 and 7, respectively, and Tb.N and Conn.D yielded one locus on chromosomes 8 and 14, respectively. We identified candidate genes with previously-reported functions in bone biology, and implicated unexpected genes whose function in bone biology has yet to be assigned. Based on the literature, among the genes that ranked particularly high in our analyses (P < 10-6) and which have a validated causal role in skeletal biology, are Avp, Oxt, B2m (associated with BV/TV), Cnot7 (with Tb.N), Pcsk6, Rgma (with Tb.Th), Rb1, and Cpb2 (with Conn.D). Other candidate genes strongly suggested by our analyses are Sgcz, Fgf20 (associated with Tb.N), and Chd2 (with Tb.Th).
We have demonstrated for the first time genome-wide significant association between several genetic loci and trabecular microstructural parameters for genes with previously reported experimental observations, as well as proposing a role for new candidate genes with no previously characterized skeletal function.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-2213-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4661944  PMID: 26611327
Trabecular bone microarchitecture; Collaborative cross; Osteoporosis; Fracture risk; Genome-wide association study; QTL mapping; Mouse genetics
14.  Applying the ARRIVE Guidelines to an In Vivo Database 
PLoS Biology  2015;13(5):e1002151.
The Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines were developed to address the lack of reproducibility in biomedical animal studies and improve the communication of research findings. While intended to guide the preparation of peer-reviewed manuscripts, the principles of transparent reporting are also fundamental for in vivo databases. Here, we describe the benefits and challenges of applying the guidelines for the International Mouse Phenotyping Consortium (IMPC), whose goal is to produce and phenotype 20,000 knockout mouse strains in a reproducible manner across ten research centres. In addition to ensuring the transparency and reproducibility of the IMPC, the solutions to the challenges of applying the ARRIVE guidelines in the context of IMPC will provide a resource to help guide similar initiatives in the future.
Transparent reporting is key to ensuring reproducibility of animal research. This article examines the challenges of applying the ARRIVE guidelines to a large-scale, collaborative, in vivo research initiative—the International Mouse Phenotyping Consortium.
PMCID: PMC4439173  PMID: 25992600
15.  Molecular Signatures of Major Depression 
Current Biology  2015;25(9):1146-1156.
Adversity, particularly in early life, can cause illness. Clues to the responsible mechanisms may lie with the discovery of molecular signatures of stress, some of which include alterations to an individual’s somatic genome. Here, using genome sequences from 11,670 women, we observed a highly significant association between a stress-related disease, major depression, and the amount of mtDNA (p = 9.00 × 10−42, odds ratio 1.33 [95% confidence interval [CI] = 1.29–1.37]) and telomere length (p = 2.84 × 10−14, odds ratio 0.85 [95% CI = 0.81–0.89]). While both telomere length and mtDNA amount were associated with adverse life events, conditional regression analyses showed the molecular changes were contingent on the depressed state. We tested this hypothesis with experiments in mice, demonstrating that stress causes both molecular changes, which are partly reversible and can be elicited by the administration of corticosterone. Together, these results demonstrate that changes in the amount of mtDNA and telomere length are consequences of stress and entering a depressed state. These findings identify increased amounts of mtDNA as a molecular marker of MD and have important implications for understanding how stress causes the disease.
•Amount of mtDNA is increased, and telomeric DNA is shortened in major depression•Both changes can be induced with stress but are contingent on the depressed state•Changes are tissue specific and in part due to glucocorticoid secretion•Changes are in part reversible and represent switches in metabolic strategy
Cai et al. found increases in mtDNA and a reduction in telomeric DNA in cases of major depression using whole-genome sequencing. Both changes are depression state dependent. Mice exposed to chronic stress or glucorticoids showed that these changes reflect switches in metabolic strategy and are tissue specific and partial reversible.
PMCID: PMC4425463  PMID: 25913401
16.  Microarray-based optimization to detect genomic deletion mutations 
Genomics Data  2014;2:53-54.
We performed array comparative genome hybridization (aCGH) analyses of five Arabidopsis thaliana mutants with genomic deletions ranging in size from 4 bp to > 5 kb. We used the Roche NimbleGen Arabidopsis CGH 3 × 720 K whole genome custom tiling array to optimize deletion detection. Details of the microarray design and hybridization data have been deposited at the NCBI GEO repository with accession number GSE55327.
PMCID: PMC4526893  PMID: 26258045
Microarray; Mutation; Deletion; Genome; CGH
17.  Impact of Temporal Variation on Design and Analysis of Mouse Knockout Phenotyping Studies 
PLoS ONE  2014;9(10):e111239.
A significant challenge facing high-throughput phenotyping of in-vivo knockout mice is ensuring phenotype calls are robust and reliable. Central to this problem is selecting an appropriate statistical analysis that models both the experimental design (the workflow and the way control mice are selected for comparison with knockout animals) and the sources of variation. Recently we proposed a mixed model suitable for small batch-oriented studies, where controls are not phenotyped concurrently with mutants. Here we evaluate this method both for its sensitivity to detect phenotypic effects and to control false positives, across a range of workflows used at mouse phenotyping centers. We found the sensitivity and control of false positives depend on the workflow. We show that the phenotypes in control mice fluctuate unexpectedly between batches and this can cause the false positive rate of phenotype calls to be inflated when only a small number of batches are tested, when the effect of knockout becomes confounded with temporal fluctuations in control mice. This effect was observed in both behavioural and physiological assays. Based on this analysis, we recommend two approaches (workflow and accompanying control strategy) and associated analyses, which would be robust, for use in high-throughput phenotyping pipelines. Our results show the importance in modelling all sources of variability in high-throughput phenotyping studies.
PMCID: PMC4208881  PMID: 25343444
18.  Susceptibility to klebsiella pneumonaie infection in collaborative cross mice is a complex trait controlled by at least three loci acting at different time points 
BMC Genomics  2014;15(1):865.
Klebsiella pneumoniae (Kp) is a bacterium causing severe pneumonia in immunocompromised hosts and is often associated with sepsis. With the rise of antibiotic resistant bacteria, there is a need for new effective and affordable control methods; understanding the genetic architecture of susceptibility to Kp will help in their development. We performed the first quantitative trait locus (QTL) mapping study of host susceptibility to Kp infection in immunocompetent Collaborative Cross mice (CC). We challenged 328 mice from 73 CC lines intraperitoneally with 104 colony forming units of Kp strain K2. Survival and body weight were monitored for 15 days post challenge. 48 of the CC lines were genotyped with 170,000 SNPs, with which we mapped QTLs.
CC lines differed significantly (P < 0.05) in mean survival time, between 1 to 15 days post infection, and broad sense heritability was 0.45. Distinct QTL were mapped at specific time points during the challenge. A QTL on chromosome 4 was found only on day 2 post infection, and QTL on chromosomes 8 and 18, only on day 8. By using the sequence variations of the eight inbred strain founders of the CC to refine QTL localization we identify several candidate genes.
Host susceptibility to Kp is a complex trait, controlled by multiple genetic factors that act sequentially during the course of infection.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-865) contains supplementary material, which is available to authorized users.
PMCID: PMC4201739  PMID: 25283706
Klebsiella pneumoniae; Mouse model; Collaborative cross mice; Host susceptibility; QTL mapping; Candidate genes
20.  Genetic Interactions with Sex Make a Relatively Small Contribution to the Heritability of Complex Traits in Mice 
PLoS ONE  2014;9(5):e96450.
The extent to which sex-specific genetic effects contribute to phenotypic variation is largely unknown. We applied a novel Bayesian method, sparse partitioning, to detect gene by sex (GxS) and gene by gene (GxG) quantitative loci (QTLs) in 1,900 outbred heterogeneous stock mice. In an analysis of 55 phenotypes, we detected 16 GxS and 6 GxG QTLs. The increase in the amount of phenotypic variance explained by models including GxS was small, ranging from 0.14% to 4.30%. We conclude that GxS rarely make a large overall contribution to the heritability of phenotypes, however there are cases where these will be individually important.
PMCID: PMC4014490  PMID: 24811081
21.  Microarray-based optimization to detect genomic deletion mutations 
Genomics Data  2014;2:53-54.
We performed array comparative genome hybridization (aCGH) analyses of five Arabidopsis thaliana mutants with genomic deletions ranging in size from 4 bp to > 5 kb. We used the Roche NimbleGen Arabidopsis CGH 3 × 720 K whole genome custom tiling array to optimize deletion detection. Details of the microarray design and hybridization data have been deposited at the NCBI GEO repository with accession number GSE55327.
PMCID: PMC4526893  PMID: 26258045
Microarray; Mutation; Deletion; Genome; CGH
22.  Genetic Control of Differential Acetylation in Diabetic Rats 
PLoS ONE  2014;9(4):e94555.
Post-translational protein modifications such as acetylation have significant regulatory roles in metabolic processes, but their relationship to both variation in gene expression and DNA sequence is unclear. We address this question in the Goto-Kakizaki (GK) rat inbred strain, a model of polygenic type 2 diabetes. Expression of the NAD-dependent deacetylase Sirtuin-3 is down-regulated in GK rats compared to normoglycemic Brown Norway (BN) rats. We show first that a promoter SNP causes down-regulation of Sirtuin-3 expression in GK rats. We then use mass-spectrometry to identify proteome-wide differential lysine acetylation of putative Sirtuin-3 protein targets in livers of GK and BN rats. These include many proteins in pathways connected to diabetes and metabolic syndrome. We finally sequence GK and BN liver transcriptomes and find that mRNA expression of these targets does not differ significantly between GK and BN rats, in contrast to other components of the same pathways. We conclude that physiological differences between GK and BN rats are mediated by a combination of differential protein acetylation and gene transcription and that genetic variation can modulate acetylation independently of expression.
PMCID: PMC3990556  PMID: 24743600
23.  Microarray-based ultra-high resolution discovery of genomic deletion mutations 
BMC Genomics  2014;15:224.
Oligonucleotide microarray-based comparative genomic hybridization (CGH) offers an attractive possible route for the rapid and cost-effective genome-wide discovery of deletion mutations. CGH typically involves comparison of the hybridization intensities of genomic DNA samples with microarray chip representations of entire genomes, and has widespread potential application in experimental research and medical diagnostics. However, the power to detect small deletions is low.
Here we use a graduated series of Arabidopsis thaliana genomic deletion mutations (of sizes ranging from 4 bp to ~5 kb) to optimize CGH-based genomic deletion detection. We show that the power to detect smaller deletions (4, 28 and 104 bp) depends upon oligonucleotide density (essentially the number of genome-representative oligonucleotides on the microarray chip), and determine the oligonucleotide spacings necessary to guarantee detection of deletions of specified size.
Our findings will enhance a wide range of research and clinical applications, and in particular will aid in the discovery of genomic deletions in the absence of a priori knowledge of their existence.
PMCID: PMC3998191  PMID: 24655320
Mutation; Deletion; Microarray; Genome; Comparative genomic hybridization; Probe density
24.  Natural Polymorphisms in Tap2 Influence Negative Selection and CD4∶CD8 Lineage Commitment in the Rat 
PLoS Genetics  2014;10(2):e1004151.
Genetic variation in the major histocompatibility complex (MHC) affects CD4∶CD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4∶CD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4∶CD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of ∼0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells.
Author Summary
Peptides from degraded cytoplasmic proteins are transported via TAP into the endoplasmic reticulum for loading onto MHC class I molecules. TAP is encoded by Tap1 and Tap2, which in rodents are located close to the MHC class I genes. In the rat, genetic variation in Tap2 gives rise to two different transporters: a promiscuous A variant (TAP-A) and a more restrictive B variant (TAP-B). It has been proposed that the class I molecule in the DA rat (RT1-Aa) has co-evolved with TAP-A and it has been shown that RT1-Aa antigenicity is changed when co-expressed with TAP-B. To study the contribution of different allelic combinations of RT1-A and Tap2 to the variation in MHC expression and T cell selection, we generated DA rats with either congenic or background alleles in the RT1-A and Tap2 loci. We found increased numbers of mature CD8SP cells in the thymus of rats which co-expressed RT1-Aa and TAP-B. This increase of CD8 cells could be explained by reduced negative selection, but did not correlate with RT1-Aa expression levels on thymic antigen presenting cells. Thus, our results identify a crucial role of the TAP and the quality of the MHC class I repertoire in regulating T cell selection.
PMCID: PMC3930506  PMID: 24586191
25.  The Architecture of Parent-of-Origin Effects in Mice 
Cell  2014;156(1-2):332-342.
The number of imprinted genes in the mammalian genome is predicted to be small, yet we show here, in a survey of 97 traits measured in outbred mice, that most phenotypes display parent-of-origin effects that are partially confounded with family structure. To address this contradiction, using reciprocal F1 crosses, we investigated the effects of knocking out two nonimprinted candidate genes, Man1a2 and H2-ab1, that reside at nonimprinted loci but that show parent-of-origin effects. We show that expression of multiple genes becomes dysregulated in a sex-, tissue-, and parent-of-origin-dependent manner. We provide evidence that nonimprinted genes can generate parent-of-origin effects by interaction with imprinted loci and deduce that the importance of the number of imprinted genes is secondary to their interactions. We propose that this gene network effect may account for some of the missing heritability seen when comparing sibling-based to population-based studies of the phenotypic effects of genetic variants.
Graphical Abstract
•Heritability of murine complex traits has a significant parent-of-origin effect•Many mouse quantitative trait loci show parent-of-origin effects•Gene knockouts induce parent-of-origin-like expression changes in reciprocal crosses
A surprisingly large proportion of traits exhibiting inheritance patterns based on parent of origin indicates a network of interactions between imprinted and nonimprinted genes. The results suggest that these interactions may account for some of the missing heritability seen when comparing sibling-based to population-based studies of the phenotypic effects of genetic variants.
PMCID: PMC3898482  PMID: 24439386

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