A wealth of genetic associations for cardiovascular and metabolic phenotypes in humans has been accumulating over the last decade, in particular a large number of loci derived from recent genome wide association studies (GWAS). True complex disease-associated loci often exert modest effects, so their delineation currently requires integration of diverse phenotypic data from large studies to ensure robust meta-analyses. We have designed a gene-centric 50 K single nucleotide polymorphism (SNP) array to assess potentially relevant loci across a range of cardiovascular, metabolic and inflammatory syndromes. The array utilizes a “cosmopolitan” tagging approach to capture the genetic diversity across ∼2,000 loci in populations represented in the HapMap and SeattleSNPs projects. The array content is informed by GWAS of vascular and inflammatory disease, expression quantitative trait loci implicated in atherosclerosis, pathway based approaches and comprehensive literature searching. The custom flexibility of the array platform facilitated interrogation of loci at differing stringencies, according to a gene prioritization strategy that allows saturation of high priority loci with a greater density of markers than the existing GWAS tools, particularly in African HapMap samples. We also demonstrate that the IBC array can be used to complement GWAS, increasing coverage in high priority CVD-related loci across all major HapMap populations. DNA from over 200,000 extensively phenotyped individuals will be genotyped with this array with a significant portion of the generated data being released into the academic domain facilitating in silico replication attempts, analyses of rare variants and cross-cohort meta-analyses in diverse populations. These datasets will also facilitate more robust secondary analyses, such as explorations with alternative genetic models, epistasis and gene-environment interactions.

A major task in dissecting the genetics of complex traits is to identify causal genes for disease phenotypes. We previously developed a method to infer causal relationships among genes through the integration of DNA variation, gene transcription, and phenotypic information. Here we validated our method through the characterization of transgenic and knockout mouse models of candidate genes that were predicted to be causal for abdominal obesity. Perturbation of eight out of the nine genes, with Gas7, Me1 and Gpx3 being novel, resulted in significant changes in obesity related traits. Liver expression signatures revealed alterations in common metabolic pathways and networks contributing to abdominal obesity and overlapped with a macrophage-enriched metabolic network module that is highly associated with metabolic traits in mice and humans. Integration of gene expression in the design and analysis of traditional F2 intercross studies allows high confidence prediction of causal genes and identification of involved pathways and networks.

We previously used high-density expression arrays to interrogate a genetic cross between strains C3H/HeJ and C57BL/6J and observed thousands of differences in gene expression between sexes. We now report analyses of the molecular basis of these sex differences and of the effects of sex on gene expression networks. We analyzed liver gene expression of hormone-treated gonadectomized mice as well as XX male and XY female mice. Differences in gene expression resulted in large part from acute effects of gonadal hormones acting in adulthood, and the effects of sex chromosomes, apart from hormones, were modest. We also determined whether there are sex differences in the organization of gene expression networks in adipose, liver, skeletal muscle, and brain tissue. Although coexpression networks of highly correlated genes were largely conserved between sexes, some exhibited striking sex dependence. We observed strong body fat and lipid correlations with sex-specific modules in adipose and liver as well as a sexually dimorphic network enriched for genes affected by gonadal hormones. Finally, our analyses identified chromosomal loci regulating sexually dimorphic networks. This study indicates that gonadal hormones play a strong role in sex differences in gene expression. In addition, it results in the identification of sex-specific gene coexpression networks related to genetic and metabolic traits.

The genetic factors contributing to the complex disorder of myocardial calcification are largely unknown. Using a mouse model, we fine-mapped the major locus (Dyscalc1) contributing to the dystrophic cardiac calcification (DCC) to an 840-kb interval containing 38 genes. We then identified the causal gene by using an approach integrating genetic segregation and expression array analyses to identify, on a global scale, cis-acting DNA variations that perturb gene expression. By studying two intercrosses, in which the DCC trait segregates, a single candidate gene (encoding the ATPbinding cassette transporter ABCC6) was identified. Transgenic complementation confirmed Abcc6 as the underlying causal gene for Dyscalc1. We demonstrate that in the cross, the expression of Abcc6 is highly correlated with the local mineralization regulatory system and the BMP2-Wnt signaling pathway known to be involved in the systemic regulation of calcification, suggesting potential pathways for the action of Abcc6 in DCC. Our results demonstrate the power of the integrative genomics in discovering causal genes and pathways underlying complex traits.expression quantitative trait locus ͉ transgenic ͉ positional cloning ͉ osteopontin C haracterized by hydroxyapatite deposition in necrotic myocytes, myocardial calcification is common in specific forms of cardiomyopathy and in myocardial infarction. It has been estimated that Ϸ8% of patients with severe myocardial infarction develop myocardial calcification within 6 years, suggesting a genetic predisposition for postinjury healing and remodeling processes (1). Historically, dystrophic cardiac calcification (DCC) has been considered a spontaneous form of cardiomyopathy in mice, associated with a variety of predisposing factors, but with normal blood levels of calcium and phosphate. Experimentally, it can be reproducibly initiated using a transdiaphragmal freeze-thaw injury or a high-phosphorous (HP) diet (2, 3). Several inbred mouse strains, including C3H/HeJ (C3H) and DBA/2J (DBA), are highly susceptible, whereas many other inbred mouse strains, including C57BL/6J (B6), C57BL/10J (B10), A/J, MRL/MpJ, and BALB/cJ are resistant (refs. 4 and 5; X.W., T.A.D., and A.J.L., unpublished data). In DCC susceptible strains, calcification has also been observed in skeletal muscle, including in the tongue and diaphragm, and kidney, suggesting a systemic defect (2, 5).Using quantitative trait locus (QTL) analysis of an F 2 intercross between B6 and C3H mice (BxH), we previously mapped four DCC loci (6, 7). The locus on chromosome 7 (Dyscalc1), which exhibits recessive inheritance, explains 31% of the total genetic variance and is the major contributor. Dyscalc1 was confirmed by separate intercrosses of B6 and DBA mice (BxD) (5, 8). The C3H strain was originally derived from an outbreeding experiment of the DBA strain, leading us to hypothesize that the susceptible strains C3H and DBA share a common diseasecausing allele (8). To fine map the Dyscalc1 locus, we screened a panel of recombinant congenic (RC) s...

Abstract-Paraoxonase 3 (PON3) is a member of the PON family, which includes PON1, PON2, and PON3. Recently, PON3 was shown to prevent the oxidation of low-density lipoprotein in vitro. To test the role of PON3 in atherosclerosis and related traits, 2 independent lines of human PON3 transgenic (Tg) mice on the C57BL/6J (B6) background were constructed. Human PON3 mRNA was detected in various tissues, including liver, lung, kidney, brain, adipose, and aorta, of both lines of Tg mice. The human PON3 mRNA levels in the livers of PON3 Tg mice were 4-to 7-fold higher as compared with the endogenous mouse Pon3 mRNA levels. Human PON3 protein and activity were detected in the livers of Tg mice as well. No significant differences in plasma total, high-density lipoprotein, and very-low-density lipoprotein/low-density lipoprotein cholesterol and triglyceride and glucose levels were observed between the PON3 Tg and non-Tg mice. Interestingly, atherosclerotic lesion areas were significantly smaller in both lines of male PON3 Tg mice as compared with the male non-Tg littermates on B6 background fed an atherogenic diet. When bred onto the low-density lipoprotein receptor knockout mouse background, the male PON3 Tg mice also exhibited decreased atherosclerotic lesion areas and decreased expression of monocyte chemoattractant protein-1 in the aorta as compared with the male non-Tg littermates. In addition, decreased adiposity and lower circulating leptin levels were observed in both lines of male PON3 Tg mice as compared with the male non-Tg mice. In an F2 cross, adipose Pon3 mRNA levels inversely correlated with adiposity and related traits. Our study demonstrates that elevated PON3 expression significantly decreases atherosclerotic lesion formation and adiposity in male mice. PON3 may play an important role in protection against obesity and atherosclerosis.

By treating the transcript abundance as a quantitative trait, gene expression can be mapped to local or distant genomic regions relative to the gene encoding the transcript. Local expression quantitative trait loci (eQTL) generally act in cis (that is, control the expression of only the contiguous structural gene), whereas distal eQTL act in trans. Distal eQTL are more difficult to identify with certainty due to the fact that significant thresholds are very high since all regions of the genome must be tested, and confounding factors such as batch effects can produce false positives. Here, we compare findings from two large genetic crosses between mouse strains C3H/HeJ and C57BL/6J to evaluate the reliability of distal eQTL detection, including ''hotspots'' influencing the expression of multiple genes in trans. We found that .63% of local eQTL and .18% of distal eQTL were replicable at a threshold of LOD . 4.3 between crosses and 76% of local and .24% of distal eQTL at a threshold of LOD . 6. Additionally, at LOD . 4.3 four tissues studied (adipose, brain, liver, and muscle) exhibited .50% preservation of local eQTL and .17% preservation of distal eQTL. We observed replicated distal eQTL hotspots between the crosses on chromosomes 9 and 17. Finally, .69% of local eQTL and .10% of distal eQTL were preserved in most tissues between sexes. We conclude that most local eQTL are highly replicable between mouse crosses, tissues, and sex as compared to distal eQTL, which exhibited modest replicability.

Abstract-We report a combined genetic and genomic analysis of atherosclerosis in a cross between the strains C3H/HeJ and C57BL/6J on a hyperlipidemic apolipoprotein E-null background. We incorporated sex and sex-by-genotype interactions into our model selection procedure to identify 10 quantitative trait loci for lesion size, revealing a level of complexity greater than previously thought. Of the known risk factors for atherosclerosis, plasma triglyceride levels and plasma glucose to insulin ratios were particularly strongly, but negatively, associated with lesion size. We performed expression array analysis for 23 574 transcripts of the livers and adipose tissues of all 334 F2 mice and identified more than 10 000 expression quantitative trait loci that either mapped to the gene encoding the transcript, implying cis regulation, or to a separate locus, implying trans-regulation. The gene expression data allowed us to identify candidate genes that mapped to the atherosclerosis quantitative trait loci and for which the expression was regulated in cis. Genes highly correlated with lesions were enriched in certain known pathways involved in lesion development, including cholesterol metabolism, mitochondrial oxidative phosphorylation, and inflammation.

Abstract-Gene targeted or transgenic mice are frequently created on one genetic background and the mutation then transferred to a second genetic background by a series of backcrosses, with selection for the mutation at each generation. This is the same experimental design as that used in the production of classical congenic strains in which a genomic region from a "donor" strain is transferred to the background of a "recipient" strain by backcrossing and selection for ten or more generations. While the strategy largely results in the replacement of the donor background with the recipient background, the region flanking the selected gene remains of donor origin. Thus, the genes in this flanking region are "passengers" that travel with the selected gene, and when comparing transgenic (or knockout) mice with non-transgenic littermates, allelic differences in the passenger genes can conceivably influence the traits of interest. It now appears that this passenger gene effect may be an important confounder in studies of cardiovascular and metabolic traits that are influenced by dozens of common variations present among inbred mouse strains. Here, we discuss the problem and some approaches to avoid it.