Large-scale genetic studies are highly dependent on efficient and scalable multiplex SNP assays. In this study, we report the development of Molecular Inversion Probe technology with four-color, single array detection, applied to large-scale genotyping of up to 12,000 SNPs per reaction. While generating 38,429 SNP assays using this technology in a population of 30 trios from the Centre d'Etude Polymorphisme Humain family panel as part of the International HapMap project, we established SNP conversion rates of ∼90% with concordance rates >99.6% and completeness levels >98% for assays multiplexed up to 12,000plex levels. Furthermore, these individual metrics can be "traded off" and, by sacrificing a small fraction of the conversion rate, the accuracy can be increased to very high levels. No loss of performance is seen when scaling from 6,000plex to 12,000plex assays, strongly validating the ability of the technology to suppress cross-reactivity at high multiplex levels. The results of this study demonstrate the suitability of this technology for comprehensive association studies that use targeted SNPs in indirect linkage disequilibrium studies or that directly screen for causative mutations.

This article is available online at http://www.jlr.org provided insight into the many different genetic factors that contribute to the disease ( 3-7 ). Using data acquired from thousands of patients and healthy controls, these studies have collectively identifi ed 35 genetic loci associated with CAD ( 8, 9 ). Although 10 of the recently identifi ed CAD risk loci work through known risk factors, such as lipids and blood pressure, this is not the case for the majority of loci ( 3 ), implying that key pathways leading to coronary atherosclerosis are yet to be discovered. Given their fi rm association with disease risk, novel CAD loci provide a solid foundation to unravel disease networks.As GWAS results do not provide functional information on the loci identifi ed, additional studies are needed to determine the candidate genes and their role in disease. The immediate challenges associated with the validation of GWAS candidate genes include identifying the likely cell types in which the risk variants and genes function, determining which of the multiple candidate genes represented by each CAD locus contribute to disease, and defi ning the functions of poorly annotated candidate genes. Expression analyses of candidate genes under defi ned conditions in model organisms and their associations with risk variants in human samples provide a powerful way to address these issues and predict the causal candidate genes.It is likely that at least some of the novel genes (that is, those not affecting known risk factors, such as plasma lipids or blood pressure) are perturbing vessel wall or infl ammatory cell functions. Endothelial cells (EC) play a critical role in the initiation and progression of atherosclerosis. Abstract Recent genome-wide association studies (GWAS)have identifi ed 35 loci that signifi cantly associate with coronary artery disease (CAD) susceptibility. The majority of the genes represented in these loci have not previously been studied in the context of atherosclerosis. To characterize the roles of these candidate genes in the vessel wall, we determined their expression levels in endothelial, smooth muscle, and macrophage cells isolated from healthy, prelesioned, and lesioned mouse aortas. We also performed expression quantitative locus (eQTL) mapping of these genes in human endothelial cells under control and proatherogenic conditions. Of the 57 genes studied, 31 were differentially expressed in one or more cell types in disease state in mice, and the expression levels of 8 were signifi cantly associated with the CAD SNPs in human cells, 7 of which were also differentially expressed in mice. By integrating human and mouse results, we predict that PPAP2B , GALNT4 , MAPKAPK5 , TCTN1 , SRR , SNF8 , and ICAM1 play a causal role in the susceptibility to atherosclerosis through a role in the vasculature. Additionally, we highlight the genetic complexity of a subset of CAD loci through the differential expression of multiple candidate genes per locus and the involvement of genes that lie outside linkage disequilib...

Summary Heparan sulfate proteoglycans (HSPGs) are an important constituent of the macrophage glycocalyx and extracellular microenvironment. To examine their role in atherogenesis, we inactivated the biosynthetic gene N-acetylglucosamine N-deacetylase-N-sulfotransferase 1 (Ndst1) in macrophages and crossbred the strain to Ldlr−/− mice. When placed on an atherogenic diet, Ldlr−/−Ndst1f/fLysMCre+ mice had increased atherosclerotic plaque area and volume compared to Ldlr−/− mice. Diminished sulfation of heparan sulfate resulted in enhanced chemokine expression, increased macrophages in plaques, increased expression of ACAT2, a key enzyme in cholesterol ester storage, and increased foam cell conversion. Motif analysis of promoters of up-regulated genes suggested increased Type I Interferon signaling, which was confirmed by elevation of STAT1 phosphorylation induced by IFN-β. The pro-inflammatory macrophages derived from Ndst1f/fLysMCre+ mice also sensitized the animals to diet-induced obesity. We propose that macrophage HSPGs control basal activation of macrophages by maintaining Type I interferon reception in a quiescent state through sequestration of IFN-β.

Background-We previously mapped a quantitative trait locus on chromosome 15 in mice contributing to high-density lipoprotein cholesterol and triglyceride levels and now report the identification of the underlying gene. Methods and Results-We first fine-mapped the locus by studying a series of congenic strains derived from the parental strains BALB/cJ and MRL/MpJ. Analysis of gene expression and sequencing followed by transgenic complementation led to the identification of zinc fingers and homeoboxes 2 (Zhx2), a transcription factor previously implicated in the developmental regulation of ␣-fetoprotein. Reduced expression of the protein in BALB/cJ mice resulted in altered hepatic transcript levels for several genes involved in lipoprotein metabolism. Most notably, the Zhx2 mutation resulted in a failure to suppress expression of lipoprotein lipase, a gene normally silenced in the adult liver, and this was normalized in BALB/cJ mice carrying the Zhx2 transgene. espite several large-scale genome-wide association studies, the genetic factors contributing to lipoprotein metabolism remain poorly understood. Thus, the loci identified thus far explain Ͻ20% of the hereditary component of lipoprotein levels, except for lipoprotein(a). [1][2][3][4][5] This is explained in part by complex gene-by-environment interactions, rare variations, and population heterogeneity. 6 Moreover, some of the novel loci contain multiple genes or genes with no known connections to lipoprotein metabolism, and establishing their functions based on human studies will be difficult. 7 Conclusions-We Clinical Perspective on p 67A complementary approach to understand the biology and genetics of lipoprotein metabolism is to study natural variations in experimental organisms such as mice and rats. 8 Although the common variations contributing to interindividual differences for complex traits are unlikely to be conserved between species, one would expect a level of conservation of the pathways involved. Unfortunately, efforts to identify genes for complex traits in mice and rats have rarely been successful. 9 A major problem has been the lack of mapping resolution in quantitative trait locus (QTL) analyses. 10 We report here the dissection of a QTL for lipoprotein levels in mice with a strategy based on the analysis of congenic and subcongenic strains, followed by screening for structural and regulatory gene variations.We previously mapped a locus on chromosome 15 (Chr.15, named Hyplip 2) contributing to complex variations in total cholesterol, high-density lipoprotein cholesterol, and triglyceride levels in a genetic cross between strains BALB/cJ and MRL/MpJ (MRL). 11 To validate the QTL mapping results, the Chr.15 region from MRL was introgressed onto the background of BALB/cJ strain. Analysis of the congenic strain, named CON15, confirmed the QTL findings and revealed a striking influence of the locus on susceptibility to diet-induced atherosclerosis. 12 Further biomedical studies of the congenic strain showed that the variation influenced clea...