Abstracts CTC-RG2018

Abstracts

    Frederick J.1, Brian S. Yandell1, Karl W. Broman2

    1Department of Statistics, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA
    2Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA

    Testing close linkage vs. pleiotropy in multiparental populations

    Multiparental populations, such as the Diversity Outbred mouse population, are a new resource for systems genetics studies. Distinguishing close linkage of distinct quantitative trait loci from one pleiotropic locus that associates with multiple traits has implications in biomedical research, plant and animal breeding, population genetics, and other genetics applications. Presence of two distinct loci potentially enables selective modification of one locus at a time. In the case of a single pleiotropic locus, it may be difficult or impossible to modify the locus without influencing both traits. We extend methods of Jiang and Zeng [1] to develop a likelihood ratio test for the alternative hypothesis of close linkage of two loci against the null hypothesis of pleiotropy for a pair of traits that map to a single genomic region. Unlike previous tests of these competing hypotheses, our test incorporates polygenic random effects to account for complex patterns of relatedness among subjects. Additionally, our test accommodates more than two founder alleles. We use a parametric bootstrap to determine statistical significance of likelihood ratio test statistics. We characterize our test’s type I error rate and power to detect close linkage of two loci in simulation studies, where we find that it is slightly conservative and has reasonable power when the univariate LOD peaks are strong. To demonstrate its practical utility, we apply our test to data from a study of 261 Diversity Outbred mice. We perform pairwise analyses of three traits that map to a single region on chromosome 8. We find evidence that there are two distinct QTL present in the region. We share our methods in a freely available software package (https://github.com/fboehm/qtl2pleio) for the R statistical computing environment.

    Astrid Gall1

    1European Molecular Biology Laboratory – European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SD

    Ensembl tools for analysis of variants in complex traits: a worked example

    The Ensembl genome browser (www.ensembl.org)1 provides visualisation and analysis of integrated genomic data, including genes, variants, comparative genomics and gene regulation, for over 100 species. This workshop provides guidance for scientists who have not yet discovered the power and depth of this resource.

    Ensembl can be used to analyse variation data, such as from whole genome variant calling, to evaluate likely candidate genes and variants involved in complex traits.

    A brief introduction to Ensembl will be followed by hands-on demonstrations and exercises:

    • Use the Ensembl Variant Effect Predictor (VEP)2 tool to predict the functional consequences of a set variants identified from sequencing a rat disease model.
    • Work through a deep dive exploration of a single variant identified from the VEP analysis, finding affected genes and transcripts, and associated phenotypes.
    • From the list of genes affected by the variant of interest, explore a single gene, exporting its sequence, homologues and GO terms.

    Participants will learn basic workflows which can be adapted to their own research questions. They will be able to learn about the wide range of data in Ensembl, and have the opportunity to think about how this data might be informative for their own research. Importantly, participants will be able to engage with the Ensembl community, finding sources of help and documentation.

    Workshop materials, including slides, demonstration screenshots, exercises and solutions will be made available before the workshop and will remain permanently online at our training portal: https://training.ensembl.org.

    Michelle N Perry1, Cynthia L Smith1, Carol J Bult1, and MGI Curation Group1

    1Jackson Laboratory, Bar Harbor, Main, USA, 04684

    Refinement of QTL candidate genes using Mouse Genome Informatics

    While QTL may span megabases of genomic sequence involving many loci, the comparison of QTL-associated phenotypes with those of known engineered and induced mutations within the genomic region offers a means for identifying candidate genes. Further refinement of candidate gene lists and sequencing targets is possible by examining strain-specific SNP likely to contribute to disruptions in gene function.

    Modern sequencing capabilities combined with the breadth of available inbred strains and characterized outbred stocks make translating phenotype to its causative sequence variation easier than ever with the use of computational tools. In addition to serving as the authoritative source for mouse gene, allele, and strain nomenclature, Mouse Genome Informatics (MGI, www.informatics.jax.org) offers an integrated database encompassing: engineered alleles and spontaneous mutations, QTL, SNP, embryonic expression patterns, Mammalian Phenotype Ontology annotations describing phenotypes, and Disease Ontology (DO) annotations for relating human diseases/syndromes to genotypes.

    As of April 2018, MGI has curated over 6,564 QTL with 12,414 strain-specific variants, nearly all of which are associated with a chromosome and many to a specific genomic sequence range. The MGI website offers a variety of query forms and quick search options to access QTL details such as mapping data and phenotypic descriptions. Bulk data access is available from downloadable reports and from MouseMine.

    There at least 26 seizure susceptibility QTL that differ between C57BL/6J and DBA/2J. Of these, audiogenic seizure prone 3 (Asp3) was mapped to Chromosome 7; 27606373-37142942 bp (GRCm38). A search in MGI for genes in this interval associated with a seizure phenotype returns four genes (Mag, Scn1b, Scl7a10 and Usf2). Of the 5,657 SNPs mapped to the Asp3 interval, one is potentially disruptive to one of these four candidate genes; rs32072976 in Slc7a10 of DBA/2J results in the missense mutation of arginine to histidine.

    The ability to use MGI to connect QTL mapping intervals with phenotypic alleles and SNPs makes it a valuable resource to identify candidate genes and regions of interest that differ between strains. MGI is currently developing strain-specific pages to summarize the known mutations, QTL, phenotypes and diseases that are characteristic of specific strains. New visualization tools are also being developed to compare genomic intervals between strains and improve identification of candidate genes based on genomic location and curated phenotype.

    Jan Silhavy1, Ondrej Kuda1, Marie Brezinova1, Vladimir Landa1, Vaclav Zidek1, Chandra Dodia2, Franziska Kreuchwig3, Laurence Balas4, Thierry Durand4, Norbert Hübner3, Aron B. Fisher2, Jan Kopecky1, Michal Pravenec1

    1Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic
    2Institute for Environmental Medicine of the Department of Physiology, University of Pennsylvania, Philadelphia, United States
    3Max-Delbrück-Center for Molecular Medicine, Berlin, Germany
    4Institut des Biomolécules Max Mousseron, CNRS, Université Montpellier, Montpellier, France

    Nrf2-mediated Antioxidant Defense and Peroxiredoxin 6 are Linked to FAHFA biosynthetic pathway

    Fatty acid esters of hydroxy fatty acids (FAHFAs) are lipid mediators with anti-diabetic and anti-inflammatory properties that are produced in white adipose tissue (WAT) via de novo lipogenesis, but their biosynthetic enzymes are unknown. Using a combination of lipidomics in WAT, QTL mapping and correlation analyses in rat BXH/HXB recombinant inbred strains, and response to oxidative stress in murine models, we elucidated the potential pathway of biosynthesis of several FAHFAs. Analysis of WAT samples identified ~160 regioisomers documenting the complexity of this lipid class. The linkage analysis highlighted several members of Nuclear factor, erythroid 2-like 2 (Nrf2)-mediated antioxidant defense system (Prdx6, Mgst1, Mgst3, Gpx7), lipid-handling proteins (Cd36, Scd6, Acnat1, Acnat2, Baat) and family of flavin containing monooxygenase (Fmo) as the positional candidate genes. Transgenic expression of Nrf2 and deletion of Prdx6 genes resulted in reduction of palmitic acid ester of 9-hydroxystearic acid (9-PAHSA) and 11-PAHSA levels, while oxidative stress induced by an inhibitor of glutathione synthesis increased PAHSA levels nonspecifically. Our results indicate that the synthesis of FAHFAs via carbohydrate-responsive element-binding protein (ChREBP)-driven de novo lipogenesis depends on the adaptive antioxidant system and suggest that FAHFAs may link activity of this system with insulin sensitivity in peripheral tissues.

    Petr Mlejnek1, Jan Silhavy1, Miroslava Simakova1, Jaroslava Trnovska2, Hana Malinska2, Ludmila Kazdova2, Michal Pravenec1

    1Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic
    2Institute for Clinical and Experimental Medicine, Prague, Czech Republic

    CD36 regulates glucose and lipid metabolism in brown adipose tissue via insulin signaling in spontaneously hypertensive rats

    Brown adipose tissue (BAT) plays an important role in lipid and glucose metabolism in rodents and possibly also in humans. Identification of genes responsible for BAT function would shed light on underlying pathophysiological mechanisms. Recently, using weighted gene co-expression network analysis (WGCNA) in the BAT from BXH/HXB recombinant inbred strains, derived from SHR (spontaneously hypertensive rat) and BN (Brown Norway) progenitors, we identified Cd36 as the hub gene of co-expression module associated with BAT relative weight. In the current study, we performed functional experiments to validate Cd36 as a quantitative trait gene responsible for BAT weight and function. SHR-Cd36 transgenic rats with wild type Cd36 gene when compared to the SHR that harbors a deletion variant of Cd36, exhibited reduced BAT weight. SHR-Cd36 BAT incubated ex vivo with glucose showed significantly increased glucose oxidation and incorporation into BAT lipids (lipogenesis) when compared to the SHR but the difference in lipogenesis was not observed when BAT was incubated in glucose together with palmitate. No significant differences between strains were detected in palmitate oxidation and incorporation into BAT lipids. Next we tested whether the increased lipogenesis in BAT is due to the effects of Cd36 on insulin signaling which is suppressed by palmitate. This possibility is supported by finding correlations between lipogenesis and expression of Irs1, Akt1, Pik3r1 and Tbc1d4 (AS160) genes involved in insulin signaling in SHR-Cd36 but not in SHR rats. These findings demonstrate that palmitate suppresses insulin signaling via Cd36. Correlation analyses in RI strains showed that increased activity of BAT was associated with amelioration of insulin resistance and hypertension. In summary, these results demonstrate important role of Cd36 gene in regulating BAT weight and function and consequentially lipid and glucose metabolism.

    Michal Pravenec1, Jan Silhavy1, Vaclav Zidek1, Vladimir Landa1, Petr Mlejnek1, Miroslava Simakova1, Jaroslava Trnovska2, Hana Malinska2, Martina Huttl2, Ludmila Kazdova2, Tomas Mracek1, Jan Kopecky1, Josef Houstek1

    1Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic
    2Institute for Clinical and Experimental Medicine, Prague, Czech Republic

    * pravenec@biomed.cas.cz

    Mutant Wars2 gene in spontaneously hypertensive rats impairs brown adipose tissue function and predisposes to visceral obesity

    Brown adipose tissue (BAT) plays an important role in lipid and glucose metabolism in rodents and possibly also in humans. Identification of genes responsible for BAT function would shed light on underlying pathophysiological mechanisms of metabolic disturbances. Recent linkage analysis in the BXH/HXB recombinant inbred (RI) strains, derived from Brown Norway (BN) and spontaneously hypertensive rats (SHR), identified 2 closely linked quantitative trait loci (QTL) associated with glucose oxidation and glucose incorporation into BAT lipids in the vicinity of Wars2 (tryptophanyl tRNA synthetase 2 (mitochondrial)) gene on chromosome 2. The SHR harbors L53F WARS2 protein variant that was associated with reduced angiogenesis and Wars2 thus represents a prominent positional candidate gene. In the current study, we validated this candidate as a quantitative trait gene (QTG) using transgenic rescue experiment. SHR-Wars2 transgenic rats with wild type Wars2 gene when compared to SHR, showed more efficient mitochondrial proteosynthesis and increased mitochondrial respiration, which was associated with increased glucose oxidation and incorporation into BAT lipids, and with reduced weight of visceral fat. Correlation analyses in RI strains showed that increased activity of BAT was associated with amelioration of insulin resistance in muscle and white adipose tissue. In summary, these results demonstrate important role of Wars2 gene in regulating BAT function and consequently lipid and glucose metabolism.

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