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1.  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.
doi:10.1371/journal.pgen.1004151
PMCID: PMC3930506  PMID: 24586191
2.  The Architecture of Parent-of-Origin Effects in Mice 
Cell  2014;156(1-2):332-342.
Summary
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
Highlights
•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.
doi:10.1016/j.cell.2013.11.043
PMCID: PMC3898482  PMID: 24439386
3.  Ablating Adult Neurogenesis in the Rat Has No Effect on Spatial Processing: Evidence from a Novel Pharmacogenetic Model 
PLoS Genetics  2013;9(9):e1003718.
The function of adult neurogenesis in the rodent brain remains unclear. Ablation of adult born neurons has yielded conflicting results about emotional and cognitive impairments. One hypothesis is that adult neurogenesis in the hippocampus enables spatial pattern separation, allowing animals to distinguish between similar stimuli. We investigated whether spatial pattern separation and other putative hippocampal functions of adult neurogenesis were altered in a novel genetic model of neurogenesis ablation in the rat. In rats engineered to express thymidine kinase (TK) from a promoter of the rat glial fibrillary acidic protein (GFAP), ganciclovir treatment reduced new neurons by 98%. GFAP-TK rats showed no significant difference from controls in spatial pattern separation on the radial maze, spatial learning in the water maze, contextual or cued fear conditioning. Meta-analysis of all published studies found no significant effects for ablation of adult neurogenesis on spatial memory, cue conditioning or ethological measures of anxiety. An effect on contextual freezing was significant at a threshold of 5% (P = 0.04), but not at a threshold corrected for multiple testing. The meta-analysis revealed remarkably high levels of heterogeneity among studies of hippocampal function. The source of this heterogeneity remains unclear and poses a challenge for studies of the function of adult neurogenesis.
Author Summary
Adult neurogenesis occurs in the rodent brain, but its function remains unclear. Current theories support the view that adult neurogenesis in the hippocampus supports pattern separation in the hippocampus, thereby allowing animals to distinguish between similar, overlapping inputs. However the effects of pharmacological, radiation and genetic ablation of adult neurogenesis on putative hippocampal functions have been inconsistent. We developed a novel genetic model to ablate adult neurogenesis in the rat. We found that we could reduce adult neurogenesis by 98%. Rats without adult neurogenesis showed no significant difference from controls in learning and memory tasks nor spatial pattern separation. We investigated the sources of heterogeneity in published results using a meta-analysis. The source of this heterogeneity remains unclear and poses a challenge for studies of the function of adult neurogenesis.
doi:10.1371/journal.pgen.1003718
PMCID: PMC3764151  PMID: 24039591
4.  Genotype is an important determinant factor of host susceptibility to periodontitis in the Collaborative Cross and inbred mouse populations 
BMC Genetics  2013;14:68.
Background
Periodontal infection (Periodontitis) is a chronic inflammatory disease, which results in the breakdown of the supporting tissues of the teeth. Previous epidemiological studies have suggested that resistance to chronic periodontitis is controlled to some extent by genetic factors of the host. The aim of this study was to determine the phenotypic response of inbred and Collaborative Cross (CC) mouse populations to periodontal bacterial challenge, using an experimental periodontitis model. In this model, mice are co-infected with Porphyromonas gingivalis and Fusobacterium nucleatum, bacterial strains associated with human periodontal disease. Six weeks following the infection, the maxillary jaws were harvested and analyzed for alveolar bone loss relative to uninfected controls, using computerized microtomography (microCT). Initially, four commercial inbred mouse strains were examined to calibrate the procedure and test for gender effects. Subsequently, we applied the same protocol to 23 lines (at inbreeding generations 10–18) from the newly developed mouse genetic reference population, the Collaborative Cross (CC) to determine heritability and genetic variation of control bone volume prior to infection (CBV, naïve bone volume around the teeth of uninfected mice), and residual bone volume (RBV, bone volume after infection) and loss of bone volume (LBV, the difference between CBV and RBV) following infection.
Results
BALB/CJ mice were highly susceptible (P<0.05) whereas DBA/2J, C57BL/6J and A/J mice were resistant. Six lines of the tested CC population were susceptible, whereas the remaining lines were resistant to alveolar bone loss. Gender effects on bone volume were tested across the four inbred and 23 CC lines, and found not to be significant. Based on ANOVA analyses, broad-sense heritabilities were statistically significant and equal to 0.4 for CBV and 0.2 for LBV.
Conclusions
The moderate heritability values indicate that the variation in host susceptibility to the disease is controlled to an appreciable extent by genetic factors. These results strongly support the possibility of using the Collaborative Cross, as well as developing dedicated F2 (resistant x susceptible inbred strains) resource populations, for future dissection of genetic factors in periodontitis.
doi:10.1186/1471-2156-14-68
PMCID: PMC3751202  PMID: 23937452
Periodontal infection; Experimental periodontitis; microCT; Collaborative cross; Genes; Heritability
5.  Causes and Consequences of Chromatin Variation between Inbred Mice 
PLoS Genetics  2013;9(6):e1003570.
Variation at regulatory elements, identified through hypersensitivity to digestion by DNase I, is believed to contribute to variation in complex traits, but the extent and consequences of this variation are poorly characterized. Analysis of terminally differentiated erythroblasts in eight inbred strains of mice identified reproducible variation at approximately 6% of DNase I hypersensitive sites (DHS). Only 30% of such variable DHS contain a sequence variant predictive of site variation. Nevertheless, sequence variants within variable DHS are more likely to be associated with complex traits than those in non-variant DHS, and variants associated with complex traits preferentially occur in variable DHS. Changes at a small proportion (less than 10%) of variable DHS are associated with changes in nearby transcriptional activity. Our results show that whilst DNA sequence variation is not the major determinant of variation in open chromatin, where such variants exist they are likely to be causal for complex traits.
Author Summary
Regulatory sites of the genome affect gene expression and complex traits, including disease susceptibility. Variable regulatory sites are potentially interesting because they are a likely cause of phenotypic variation, providing a bridge between sequence and transcriptional variation. In this paper we identify regions of the genome where DNA is not wrapped up in chromatin (hence potentially regulatory) in eight inbred strains of mice. We compare sites that vary among strains and compare them to non-variable sites. We show that more than half of variable sites cannot be attributed to local sequence variation. Functional consequences (in terms of readily detectable changes in gene expression) are associated with less than 10% of variable DNase I hypersensitive sites. We show that variable sites are enriched for sequence variants contributing to complex traits in mice.
doi:10.1371/journal.pgen.1003570
PMCID: PMC3681629  PMID: 23785304
6.  Robust and Sensitive Analysis of Mouse Knockout Phenotypes 
PLoS ONE  2012;7(12):e52410.
A significant challenge of in-vivo studies is the identification of phenotypes with a method that is robust and reliable. The challenge arises from practical issues that lead to experimental designs which are not ideal. Breeding issues, particularly in the presence of fertility or fecundity problems, frequently lead to data being collected in multiple batches. This problem is acute in high throughput phenotyping programs. In addition, in a high throughput environment operational issues lead to controls not being measured on the same day as knockouts. We highlight how application of traditional methods, such as a Student’s t-Test or a 2-way ANOVA, in these situations give flawed results and should not be used. We explore the use of mixed models using worked examples from Sanger Mouse Genome Project focusing on Dual-Energy X-Ray Absorptiometry data for the analysis of mouse knockout data and compare to a reference range approach. We show that mixed model analysis is more sensitive and less prone to artefacts allowing the discovery of subtle quantitative phenotypes essential for correlating a gene’s function to human disease. We demonstrate how a mixed model approach has the additional advantage of being able to include covariates, such as body weight, to separate effect of genotype from these covariates. This is a particular issue in knockout studies, where body weight is a common phenotype and will enhance the precision of assigning phenotypes and the subsequent selection of lines for secondary phenotyping. The use of mixed models with in-vivo studies has value not only in improving the quality and sensitivity of the data analysis but also ethically as a method suitable for small batches which reduces the breeding burden of a colony. This will reduce the use of animals, increase throughput, and decrease cost whilst improving the quality and depth of knowledge gained.
doi:10.1371/journal.pone.0052410
PMCID: PMC3530558  PMID: 23300663
7.  Cofilin-1: A Modulator of Anxiety in Mice 
PLoS Genetics  2012;8(10):e1002970.
The genes involved in conferring susceptibility to anxiety remain obscure. We developed a new method to identify genes at quantitative trait loci (QTLs) in a population of heterogeneous stock mice descended from known progenitor strains. QTLs were partitioned into intervals that can be summarized by a single phylogenetic tree among progenitors and intervals tested for consistency with alleles influencing anxiety at each QTL. By searching for common Gene Ontology functions in candidate genes positioned within those intervals, we identified actin depolymerizing factors (ADFs), including cofilin-1 (Cfl1), as genes involved in regulating anxiety in mice. There was no enrichment for function in the totality of genes under each QTL, indicating the importance of phylogenetic filtering. We confirmed experimentally that forebrain-specific inactivation of Cfl1 decreased anxiety in knockout mice. Our results indicate that similarity of function of mammalian genes can be used to recognize key genetic regulators of anxiety and potentially of other emotional behaviours.
Author Summary
Thousands of small effect loci are believed to contribute to behavioural variation in mammals. Their abundance and small size frustrate gene identification and make it difficult to know which among them are central to the responsible biological mechanisms. Using imputed genome sequences from 2,000 outbred mice and by testing for an enrichment of functional annotations, we identify 167 candidate genes involved in anxiety. Unexpectedly, annotations implicate actin depolymerizing factors (ADFs), including cofilin-1 (Cfl1), as being involved with the expression of anxiety phenotypes in mice. We confirmed that forebrain-specific inactivation of Cfl1 decreased anxiety in knockout mice.
doi:10.1371/journal.pgen.1002970
PMCID: PMC3464202  PMID: 23055942
8.  Sequence based characterization of structural variation in the mouse genome 
Nature  2011;477(7364):326-329.
Structural variation is widespread in mammalian genomes1,2 and is an important cause of disease3, but just how abundant and important structural variants (SVs) are in shaping phenotypic variation remains unclear4,5. Without knowing how many SVs there are, and how they arise, it is difficult to discover what they do. Combining experimental with automated analyses, we identified 0.71M SVs at 0.28M sites in the genomes of thirteen classical and four wild-derived inbred mouse strains. The majority of SVs are less than 1 kilobase in size and 98% are deletions or insertions. The breakpoints of 0.16M SVs were mapped to base pair resolution allowing us to infer that insertion of retrotransposons causes more than half of SVs. Yet, despite their prevalence, SVs are less likely than other sequence variants to cause gene-expression or quantitative phenotypic variation. We identified 24 SVs that disrupt coding exons, acting as rare variants of large effect on gene function. One third of the genes so affected have immunological functions.
doi:10.1038/nature10432
PMCID: PMC3428933  PMID: 21921916
9.  Status and access to the Collaborative Cross population 
Mammalian Genome  2012;23:706-712.
The Collaborative Cross (CC) is a panel of recombinant inbred lines derived from eight genetically diverse laboratory inbred strains. Recently, the genetic architecture of the CC population was reported based on the genotype of a single male per line, and other publications reported incompletely inbred CC mice that have been used to map a variety of traits. The three breeding sites, in the US, Israel, and Australia, are actively collaborating to accelerate the inbreeding process through marker-assisted inbreeding and to expedite community access of CC lines deemed to have reached defined thresholds of inbreeding. Plans are now being developed to provide access to this novel genetic reference population through distribution centers. Here we provide a description of the distribution efforts by the University of North Carolina Systems Genetics Core, Tel Aviv University, Israel and the University of Western Australia.
doi:10.1007/s00335-012-9410-6
PMCID: PMC3463789  PMID: 22847377
10.  Mouse genomic variation and its effect on phenotypes and gene regulation 
Nature  2011;477(7364):289-294.
We report genome sequences of 17 inbred strains of laboratory mice and identify almost ten times more variants than previously known. We use these genomes to explore the phylogenetic history of the laboratory mouse and to examine the functional consequences of allele-specific variation on transcript abundance, revealing that at least 12% of transcripts show a significant tissue-specific expression bias. By identifying candidate functional variants at 718 quantitative trait loci we show that the molecular nature of functional variants and their position relative to genes vary according to the effect size of the locus. These sequences provide a starting point for a new era in the functional analysis of a key model organism.
doi:10.1038/nature10413
PMCID: PMC3276836  PMID: 21921910
11.  Regenerant Arabidopsis Lineages Display a Distinct Genome-Wide Spectrum of Mutations Conferring Variant Phenotypes 
Current Biology  2011;21(16):1385-1390.
Summary
Multicellular organisms can be regenerated from totipotent differentiated somatic cell or nuclear founders [1–3]. Organisms regenerated from clonally related isogenic founders might a priori have been expected to be phenotypically invariant. However, clonal regenerant animals display variant phenotypes caused by defective epigenetic reprogramming of gene expression [2], and clonal regenerant plants exhibit poorly understood heritable phenotypic (“somaclonal”) variation [4–7]. Here we show that somaclonal variation in regenerant Arabidopsis lineages is associated with genome-wide elevation in DNA sequence mutation rate. We also show that regenerant mutations comprise a distinctive molecular spectrum of base substitutions, insertions, and deletions that probably results from decreased DNA repair fidelity. Finally, we show that while regenerant base substitutions are a likely major genetic cause of the somaclonal variation of regenerant Arabidopsis lineages, transposon movement is unlikely to contribute substantially to that variation. We conclude that the phenotypic variation of regenerant plants, unlike that of regenerant animals, is substantially due to DNA sequence mutation.
Highlights
► Regenerant Arabidopsis lineages display heritable phenotypic variation ► Regenerant Arabidopsis lineages display elevated genome-wide DNA sequence mutation ► Regenerant DNA sequence mutations comprise a distinct molecular spectrum ► Regenerant base substitution mutations confer heritable phenotypic variation
doi:10.1016/j.cub.2011.07.002
PMCID: PMC3162137  PMID: 21802297
12.  Bioinformatics tools and database resources for systems genetics analysis in mice—a short review and an evaluation of future needs 
Briefings in Bioinformatics  2011;13(2):135-142.
During a meeting of the SYSGENET working group ‘Bioinformatics’, currently available software tools and databases for systems genetics in mice were reviewed and the needs for future developments discussed. The group evaluated interoperability and performed initial feasibility studies. To aid future compatibility of software and exchange of already developed software modules, a strong recommendation was made by the group to integrate HAPPY and R/qtl analysis toolboxes, GeneNetwork and XGAP database platforms, and TIQS and xQTL processing platforms. R should be used as the principal computer language for QTL data analysis in all platforms and a ‘cloud’ should be used for software dissemination to the community. Furthermore, the working group recommended that all data models and software source code should be made visible in public repositories to allow a coordinated effort on the use of common data structures and file formats.
doi:10.1093/bib/bbr026
PMCID: PMC3294237  PMID: 22396485
QTL mapping; database; mouse; systems genetics
13.  Genetic Architecture of Flowering-Time Variation in Arabidopsis thaliana 
Genetics  2011;188(2):421-433.
The onset of flowering is an important adaptive trait in plants. The small ephemeral species Arabidopsis thaliana grows under a wide range of temperature and day-length conditions across much of the Northern hemisphere, and a number of flowering-time loci that vary between different accessions have been identified before. However, only few studies have addressed the species-wide genetic architecture of flowering-time control. We have taken advantage of a set of 18 distinct accessions that present much of the common genetic diversity of A. thaliana and mapped quantitative trait loci (QTL) for flowering time in 17 F2 populations derived from these parents. We found that the majority of flowering-time QTL cluster in as few as five genomic regions, which include the locations of the entire FLC/MAF clade of transcription factor genes. By comparing effects across shared parents, we conclude that in several cases there might be an allelic series caused by rare alleles. While this finding parallels results obtained for maize, in contrast to maize much of the variation in flowering time in A. thaliana appears to be due to large-effect alleles.
doi:10.1534/genetics.111.126607
PMCID: PMC3122318  PMID: 21406681
15.  A Genetic and Functional Relationship between T Cells and Cellular Proliferation in the Adult Hippocampus 
PLoS Biology  2010;8(12):e1000561.
A large correlation between variation in T cell subsets and hippocampal neurogenesis suggests that the immune system has an unexpectedly large influence on the brain.
Neurogenesis continues through the adult life of mice in the subgranular zone of the dentate gyrus in the hippocampus, but its function remains unclear. Measuring cellular proliferation in the hippocampus of 719 outbred heterogeneous stock mice revealed a highly significant correlation with the proportions of CD8+ versus CD4+ T lymphocyte subsets. This correlation reflected shared genetic loci, with the exception of the H-2Ea locus that had a dominant influence on T cell subsets but no impact on neurogenesis. Analysis of knockouts and repopulation of TCRα-deficient mice by subsets of T cells confirmed the influence of T cells on adult neurogenesis, indicating that CD4+ T cells or subpopulations thereof mediate the effect. Our results reveal an organismal impact, broader than hitherto suspected, of the natural genetic variation that controls T cell development and homeostasis.
Author Summary
In adult mice new neurons are produced in the hippocampus, where they are thought to influence learning, memory, and emotional regulation. The mechanisms and functions of this neurogenesis, however, remain unclear. Here we report that in different strains of mice, variation in cellular proliferation in the hippocampus (an index of neurogenesis) correlates with variation in the relative proportions of the ratio of CD4+ to CD8+ T cells (an immunology phenotype). We also show that T cells can influence neurogenesis (but that neurogenesis does not influence T cells) by analyzing knockouts, depleting mice of T cells, and repopulating alymphoid animals. The strong genetic correlation between T cells and cellular proliferation in the hippocampus contrasts with the weak, often non-significant, correlation with behavioral phenotypes. Of significance, the findings here suggest that modulation of the functions of the hippocampus to influence behavior is not the primary role of neurogenesis.
doi:10.1371/journal.pbio.1000561
PMCID: PMC3001898  PMID: 21179499
16.  Elusive Copy Number Variation in the Mouse Genome 
PLoS ONE  2010;5(9):e12839.
Background
Array comparative genomic hybridization (aCGH) to detect copy number variants (CNVs) in mammalian genomes has led to a growing awareness of the potential importance of this category of sequence variation as a cause of phenotypic variation. Yet there are large discrepancies between studies, so that the extent of the genome affected by CNVs is unknown. We combined molecular and aCGH analyses of CNVs in inbred mouse strains to investigate this question.
Principal Findings
Using a 2.1 million probe array we identified 1,477 deletions and 499 gains in 7 inbred mouse strains. Molecular characterization indicated that approximately one third of the CNVs detected by the array were false positives and we estimate the false negative rate to be more than 50%. We show that low concordance between studies is largely due to the molecular nature of CNVs, many of which consist of a series of smaller deletions and gains interspersed by regions where the DNA copy number is normal.
Conclusions
Our results indicate that CNVs detected by arrays may be the coincidental co-localization of smaller CNVs, whose presence is more likely to perturb an aCGH hybridization profile than the effect of an isolated, small, copy number alteration. Our findings help explain the hitherto unexplored discrepancies between array-based studies of copy number variation in the mouse genome.
doi:10.1371/journal.pone.0012839
PMCID: PMC2943477  PMID: 20877625
17.  Commercially Available Outbred Mice for Genome-Wide Association Studies 
PLoS Genetics  2010;6(9):e1001085.
Genome-wide association studies using commercially available outbred mice can detect genes involved in phenotypes of biomedical interest. Useful populations need high-frequency alleles to ensure high power to detect quantitative trait loci (QTLs), low linkage disequilibrium between markers to obtain accurate mapping resolution, and an absence of population structure to prevent false positive associations. We surveyed 66 colonies for inbreeding, genetic diversity, and linkage disequilibrium, and we demonstrate that some have haplotype blocks of less than 100 Kb, enabling gene-level mapping resolution. The same alleles contribute to variation in different colonies, so that when mapping progress stalls in one, another can be used in its stead. Colonies are genetically diverse: 45% of the total genetic variation is attributable to differences between colonies. However, quantitative differences in allele frequencies, rather than the existence of private alleles, are responsible for these population differences. The colonies derive from a limited pool of ancestral haplotypes resembling those found in inbred strains: over 95% of sequence variants segregating in outbred populations are found in inbred strains. Consequently it is possible to impute the sequence of any mouse from a dense SNP map combined with inbred strain sequence data, which opens up the possibility of cataloguing and testing all variants for association, a situation that has so far eluded studies in completely outbred populations. We demonstrate the colonies' potential by identifying a deletion in the promoter of H2-Ea as the molecular change that strongly contributes to setting the ratio of CD4+ and CD8+ lymphocytes.
Author Summary
We show that commercially available mice are a resource for detecting single genes by genome-wide association. We surveyed 66 populations and identified those with properties conducive to high-resolution mapping. Importantly, we show that the same alleles contribute to variation in different colonies, so that when mapping progress stalls in one colony, another can be used in its stead. As a proof of principle, we detect the same QTL in different colonies influencing CD4+/CD8+ ratios and refine this mapping to the gene level. We show that a deletion in the promoter of H2-Ea is the molecular change that strongly contributes to setting the ratio of CD4+ and CD8+ lymphocytes. Our results make it possible for geneticists to make informed choices on the use of colonies for genome-wide association studies of complex traits in mice.
doi:10.1371/journal.pgen.1001085
PMCID: PMC2932682  PMID: 20838427
18.  The 1001 Genomes Project for Arabidopsis thaliana 
Genome Biology  2009;10(5):107.
The 1001 Genomes project for Arabidopsis thaliana could provide an enormous boost for plant research for a modest financial investment.
We advocate here a 1001 Genomes project for Arabidopsis thaliana, the workhorse of plant genetics, which will provide an enormous boost for plant research with a modest financial investment.
doi:10.1186/gb-2009-10-5-107
PMCID: PMC2718507  PMID: 19519932
19.  GLIDERS - A web-based search engine for genome-wide linkage disequilibrium between HapMap SNPs 
BMC Bioinformatics  2009;10:367.
Background
A number of tools for the examination of linkage disequilibrium (LD) patterns between nearby alleles exist, but none are available for quickly and easily investigating LD at longer ranges (>500 kb). We have developed a web-based query tool (GLIDERS: Genome-wide LInkage DisEquilibrium Repository and Search engine) that enables the retrieval of pairwise associations with r2 ≥ 0.3 across the human genome for any SNP genotyped within HapMap phase 2 and 3, regardless of distance between the markers.
Description
GLIDERS is an easy to use web tool that only requires the user to enter rs numbers of SNPs they want to retrieve genome-wide LD for (both nearby and long-range). The intuitive web interface handles both manual entry of SNP IDs as well as allowing users to upload files of SNP IDs. The user can limit the resulting inter SNP associations with easy to use menu options. These include MAF limit (5-45%), distance limits between SNPs (minimum and maximum), r2 (0.3 to 1), HapMap population sample (CEU, YRI and JPT+CHB combined) and HapMap build/release. All resulting genome-wide inter-SNP associations are displayed on a single output page, which has a link to a downloadable tab delimited text file.
Conclusion
GLIDERS is a quick and easy way to retrieve genome-wide inter-SNP associations and to explore LD patterns for any number of SNPs of interest. GLIDERS can be useful in identifying SNPs with long-range LD. This can highlight mis-mapping or other potential association signal localisation problems.
doi:10.1186/1471-2105-10-367
PMCID: PMC2777181  PMID: 19878600
20.  Dynamic and Physical Clustering of Gene Expression during Epidermal Barrier Formation in Differentiating Keratinocytes 
PLoS ONE  2009;4(10):e7651.
The mammalian epidermis is a continually renewing structure that provides the interface between the organism and an innately hostile environment. The keratinocyte is its principal cell. Keratinocyte proteins form a physical epithelial barrier, protect against microbial damage, and prepare immune responses to danger. Epithelial immunity is disordered in many common diseases and disordered epithelial differentiation underlies many cancers. In order to identify the genes that mediate epithelial development we used a tissue model of the skin derived from primary human keratinocytes. We measured global gene expression in triplicate at five times over the ten days that the keratinocytes took to fully differentiate. We identified 1282 gene transcripts that significantly changed during differentiation (false discovery rate <0.01%). We robustly grouped these transcripts by K-means clustering into modules with distinct temporal expression patterns, shared regulatory motifs, and biological functions. We found a striking cluster of late expressed genes that form the structural and innate immune defences of the epithelial barrier. Gene Ontology analyses showed that undifferentiated keratinocytes were characterised by genes for motility and the adaptive immune response. We systematically identified calcium-binding genes, which may operate with the epidermal calcium gradient to control keratinocyte division during skin repair. The results provide multiple novel insights into keratinocyte biology, in particular providing a comprehensive list of known and previously unrecognised major components of the epidermal barrier. The findings provide a reference for subsequent understanding of how the barrier functions in health and disease.
doi:10.1371/journal.pone.0007651
PMCID: PMC2766255  PMID: 19888454
21.  Differential sensitivity of mouse strains to an N-alkylated imino sugar: glycosphingolipid metabolism and acrosome formation 
Pharmacogenomics  2008;9(6):717-731.
This review deals with the pharmacological properties of an alkylated monosaccharide mimetic, N-butyldeoxynojirimycin (NB-DNJ). This compound is of pharmacogenetic interest because one of its biological effects in mice – impairment of spermatogenesis, leading to male infertility – depends greatly on the genetic background of the animal. In susceptible mice, administration of NB-DNJ perturbs the formation of an organelle, the acrosome, in early post-meiotic male germ cells. In all recipient mice, irrespective of reproductive phenotype, NB-DNJ has a similar biochemical effect: inhibition of the glucosylceramidase β-glucosidase 2 and subsequent elevation of glucosylceramide, a glycosphingolipid. The questions that we now need to address are: how can glucosylceramide specifically affect early acrosome formation, and why is this contingent on genetic factors? Here we discuss relevant aspects of reproductive biology, the metabolism and cell biology of sphingolipids, and complex trait analysis; we also present a speculative model that takes our observations into account.
doi:10.2217/14622416.9.6.717
PMCID: PMC2749735  PMID: 18518850
acrosome; glucosylceramide; glycosphingolipid; imino sugar; semen parameters; sperm morphology; spermatid; spermatogenesis
22.  A Multiparent Advanced Generation Inter-Cross to Fine-Map Quantitative Traits in Arabidopsis thaliana 
PLoS Genetics  2009;5(7):e1000551.
Identifying natural allelic variation that underlies quantitative trait variation remains a fundamental problem in genetics. Most studies have employed either simple synthetic populations with restricted allelic variation or performed association mapping on a sample of naturally occurring haplotypes. Both of these approaches have some limitations, therefore alternative resources for the genetic dissection of complex traits continue to be sought. Here we describe one such alternative, the Multiparent Advanced Generation Inter-Cross (MAGIC). This approach is expected to improve the precision with which QTL can be mapped, improving the outlook for QTL cloning. Here, we present the first panel of MAGIC lines developed: a set of 527 recombinant inbred lines (RILs) descended from a heterogeneous stock of 19 intermated accessions of the plant Arabidopsis thaliana. These lines and the 19 founders were genotyped with 1,260 single nucleotide polymorphisms and phenotyped for development-related traits. Analytical methods were developed to fine-map quantitative trait loci (QTL) in the MAGIC lines by reconstructing the genome of each line as a mosaic of the founders. We show by simulation that QTL explaining 10% of the phenotypic variance will be detected in most situations with an average mapping error of about 300 kb, and that if the number of lines were doubled the mapping error would be under 200 kb. We also show how the power to detect a QTL and the mapping accuracy vary, depending on QTL location. We demonstrate the utility of this new mapping population by mapping several known QTL with high precision and by finding novel QTL for germination data and bolting time. Our results provide strong support for similar ongoing efforts to produce MAGIC lines in other organisms.
Author Summary
Most traits of economic and evolutionary interest vary quantitatively and have multiple genes affecting their expression. Dissecting the genetic basis of such traits is crucial for the improvement of crops and management of diseases. Here, we develop a new resource to identify genes underlying such quantitative traits in Arabidopsis thaliana, a genetic model organism in plants. We show that using a large population of inbred lines derived from intercrossing 19 parents, we can localize the genes underlying quantitative traits better than with existing methods. Using these lines, we were able to replicate the identification of previously known genes that affect developmental traits in A. thaliana and identify some new ones. This paper also presents all the necessary biological and computational material necessary for the scientific community to use these lines in their own research. Our results suggest that the use of lines derived from a multiparent advanced generation inter-cross (MAGIC lines) should be very useful in other organisms.
doi:10.1371/journal.pgen.1000551
PMCID: PMC2700969  PMID: 19593375
23.  Prospects for complex trait analysis in the mouse 
Mammalian Genome  2008;19(5):306-308.
doi:10.1007/s00335-008-9110-4
PMCID: PMC2515547  PMID: 18493822
25.  Finding the molecular basis of complex genetic variation in humans and mice 
I survey the state of the art in complex trait analysis, including the use of new experimental and computational technologies and resources becoming available, and the challenges facing us. I also discuss how the prospects of rodent model systems compare with association mapping in humans.
doi:10.1098/rstb.2005.1798
PMCID: PMC1609339  PMID: 16524828
complex traits; genetic mapping; association; human genetics; mouse genetics; quantitative trait locus

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